Synthesis of precursors of the rare 3-O-methyl mannose … · 2015. 12. 12. · Synthesis of...

I

André Oliveira Sequeira

Licenciado em Bioquímica

Synthesis of precursors of the rare

3-O-methylmannose polysaccharides

present in Nontuberculous

Mycobacteria

Dissertação para obtenção do Grau de Mestre em

Química Bioorgânica

Orientadora: Rita Ventura, Dra., Instituto de Tecnologia

Química e Biológica António Xavier

Co-orientadora: Teresa Barros, Prof. Dra., Faculdade de

Ciências e Tecnologia da Universidade Nova de Lisboa

Júri:

Presidente: Prof. Doutora Paula Cristina de Sério Branco

Arguente: Doutora Krasimira Todorova Markova-Petrova

Outubro de 2015

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III

André Oliveira Sequeira

Licenciado em Bioquímica

Synthesis of precursors of the rare

3-O-methylmannose polysaccharides

present in Nontuberculous

Mycobacteria

Dissertação para obtenção do Grau de Mestre em

Química Bioorgânica

Orientadora: Maria Rita Ventura, Dra., Instituto de

Tecnologia Química e Biológica António Xavier

Co-orientadora: Teresa Barros, Prof. Dra., Faculdade de

Ciências e Tecnologia da Universidade Nova de Lisboa

Júri:

Presidente: Prof. Doutora Paula Cristina de Sério Branco

Arguente: Doutora Krasimira Todorova Markova-Petrova

Outubro de 2015

II

III

Copyrights

A Faculdade de Ciências e Tecnologia e a Universidade Nova de Lisboa têm o direito, perpétuo

e sem limites geográficos, de arquivar e publicar esta dissertação através de exemplares

impressos reproduzidos em papel ou de forma digital, ou por qualquer outro meio conhecido ou

que venha a ser inventado, e de a divulgar através de repositórios científicos e de admitir a sua

cópia e distribuição com objectivos educacionais ou de investigação, não comerciais, desde que

seja dado crédito ao autor e editor.

IV

V

Agradecimentos

Em primeiro lugar, queria agradecer à minha orientadora Dra. Rita Ventura, pelo apoio

manifestado, pela confiança, disponibilidade e pela oportunidade que me deu para realizar este

projecto. Este último ano adquiri muita experiência e foi possível adaptar-me ao ambiente que

se vive num laboratório de investigação.

Ao professor Christopher Maycock, por todo o apoio, vontade para me ajudar e a

sabedoria que transmitiu na resolução de obstáculos.

Às minhas colegas de laboratório Eva e Vanessa pela boa disposição, pelo fantástico

total apoio que me deram durante este ano e também pela paciência e que tiveram para me

passarem os seus conhecimentos.

Aos meus restantes colegas de laboratório Osvaldo, Saúl, Jessica, João, Patrícia e Chan,

pela boa disposição e pela ajuda e apoio que me deram ao longo de este ano.

Aos meus amigos da FCUL, por todos os momentos passados durante estes 5 anos, por

todo apoio que me deram neste último ano, por estarem lá quando precisava, por encontrarem

sempre forma para desanuviar. Felizmente como estávamos todos no mesmo barco foi fácil para

mim retribuir-vos o apoio. Que venham mais 5 anos.

Aos meus amigos Fctenses, que conheci através deste Mestrado, pelos momentos

divertidos e bem passados. Um especial agradecimento à Margarida (ela adora que eu a chame

assim) pelas palavras certas a dizer, por estares lá sempre presente e por ouvires as boas e as

más noticias que aconteceram este ano.

Aos meus amigos pré faculdade por me terem acompanhado desde o início desta

jornada e por estarem lá sempre quando precisar. Um especial agradecimento ao Filipe Mealha,

ao Pipo, ao Evani e ao Luís por as coisas continuarem iguais, mesmo depois de tantos anos. Que

venham mais 10 anos.

À minha família, por todo apoio nesta fase, com destaque aos meus tios, que me deram

uma grande ajuda nesta última fase do ano de dissertação.

Ao meu Pai e à minha Mãe, que nunca desistiram de mim e me deram todas as

condições para chegar onde cheguei. Pelo apoio incondicional, incentivo e apoio nos obstáculos

que foram surgindo. Espero conseguir um dia retribuir-lhes o favor. Sem vocês tudo isto não era

possível.

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Abstract

3-O-methylmannose polysaccharides (MMPs) are cytoplasmic carbohydrates

synthesized by mycobacteria, which play important intracellular roles, such as for example in

metabolism regulation. An important way to confirm if the inhibition of the synthesis of these

polysaccharides will critically affect the survival of mycobacteria is the study of the

biosynthetic pathways from these molecules on these microorganisms.

The purpose of this work is the efficient synthesis of three saccharides, which are rare

cellular precursors from the biosynthesis of the mycobacterial polysaccharides, allowing its

study. In order to obtain these molecules, a chemical strategy to connect two precursors was

used. This process is called chemical glycosylation and its importance will be highlighted as an

important alternative to enzymatic glycosylation.

The first objective was the synthesis of the disaccharides Methyl (3-O-methyl-α-D-

mannopyranosyl)-(1→4)-3-O-methyl-α-D-mannopyranoside and (3-O-Methyl-α-D-mannopyra-

nosyl)-(1→4)-3-O-methyl-(α/β)-D-mannopyranose. The mannose precursors were prepared

before the glycosylation reaction. The same mannosyl donor was used in the preparation of both

molecules and its efficient synthesis was achieved using a 8 step synthetic route from D-

mannose. A different mannosyl acceptor was used in the synthesis of each disaccharide and

their syntheses were also efficient, the first one a 4 step synthetic route from α-methyl-D-

mannose and the second one as an intermediate from the synthesis of the mannosyl donor. The

stereoselective preparation of these disaccharides was performed successfully.

The second and last objective of the proposed work was the synthesis of the

tetrasaccharide methyl (3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyra-

nosyl-(1→4)-3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyranoside. The

disaccharide acceptor and donor to be linked through a stereoselective glycosidic reaction had to

be first synthesized. Several synthetic strategies were studied. Neither the precursors nor the

tetrasaccharide were synthesized, but a final promising synthetic route for its preparation has

been proposed.

VIII

Keywords

MMPs

Saccharides

Precursors

Chemical glycosylation

IX

Resumo

Os Polissacáridos de 3-O-metil-manose (PMMs) são açúcares citoplasmáticos

sintetizados pelas micobactérias, que desempenham funções intracelulares importantes, como

por exemplo na regulação do metabolismo. Uma maneira importante de confirmar se a inibição

da síntese destes polissacáridos vai afectar criticamente a sobrevivência das micobactérias é o

estudo das vias biossintéticas destas moléculas nestes microrganismos.

O objectivo deste trabalho é a síntese eficiente de três sacáridos, que são precursores

celulares raros da biossíntese dos polissacáridos das micobactérias, permitindo o seu estudo. De

forma a se obter estas moléculas, uma estratégia química para ligar dois precursores foi usada.

Este processo é denominado de glicosilação química e a sua importância vai ser destacada como

uma importante alternativa à glicosilação enzimática.

O primeiro objectivo foi a síntese dos dissacáridos Metil-(3-O-metil-α-D-

manopiranosil)-(1→4)-3-O-metil-α-D-manopiranosídeo e (3-O-Metil-α-D-manopiranosil)-

(1→4)-3-O-metil-(α/β)-D-manopiranose. Os precursores da manose foram preparados antes da

reacção de glicosilação. O mesmo doador de manosil foi usado na preparação de ambas as

moléculas e a sua síntese eficiente foi alcançada usando uma estratégia com 8 passos, a partir da

D-manose. Um diferente aceitador manosil foi usado na preparação de cada dissacárido e as

suas sínteses foram também eficientes, o primeiro foi obtido de uma estratégia de síntese de 4

passos a partir da α-metil-manose e o segundo como um intermediário da síntese do doador

manosil. A preparação estereoselectiva destes dissacáridos foi realizada com sucesso.

O segundo e último objectivo do trabalho proposto foi a síntese do tetrassacárido Metil-

-3-O-metil-α-D-manopiranosil-(1→4)-3-O-metil-α-D-manopiranosil-(1→4)-3-O-metil-α-D-

-manopiranosil-(1→4)-3-O-metil-α-D-manopiranosídeo. Os dissacáridos aceitador e doador a

serem ligados por uma ligação glicosídica estereoselectiva tinham de ser sintetizados primeiro.

Algumas estratégias de síntese foram estudadas. Nem os percursores nem o tetrassacárido final

foram sintetizados, mas uma estratégia de síntese promissora para a sua formação foi proposta.

X

Palavras-Chave

PMMs

Sacáridos

Precursores

Glicosilação química

XI

Table of Contents

1. Introduction .................................................................................................................. 3

1.1 Location and biological function of MMPs ........................................................... 3

1.2 Chemical synthesis of mannose oligosaccharides................................................ 4

1.2.1 Mechanism ................................................................................................................... 6

2. Results and discussion ....................................................................................... 13

2.1 Disaccharide synthesis ............................................................................................... 13

2.1.1 Monosaccharide glycosyl donor synthesis ................................................................. 13

2.1.2 Monosaccharide glycosyl acceptors synthesis ........................................................... 21

2.1.3 Glycosylation reaction and hydroxyl group deprotection .......................................... 24

2.2 Tetrasaccharide synthesis ......................................................................................... 30

2.2.1 Disaccharide glycosyl acceptor synthesis .................................................................. 31

2.2.2 Disaccharide glycosyl donor synthesis ....................................................................... 48

3. Conclusion ................................................................................................................... 51

4. Experimental part ................................................................................................. 57

4.1 General conditions ...................................................................................................... 57

4.2 Solvent and Reagent Purification ........................................................................... 57

4.3 Compound list .............................................................................................................. 59

4.4 Experimental Procedures .......................................................................................... 65

5. References .................................................................................................................... 95

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XIII

List of Tables

Table 2.1 – Summary of the different experimental conditions used for the optimization of the

synthesis of 27. ............................................................................................................................ 39

Table 4.1 - Summary table of the synthesized compounds. ...................................................... 59

List of Schemes

Scheme 1.1 - The chemical glycosylation reaction between two monosaccharides. .................... 5

Scheme 1.2 – Hydrolysis of the glycosyl donor. .......................................................................... 5

Scheme 1.3 – Departure of the leaving group and formation of the oxonium ion. ....................... 6

Scheme 1.4 – Acetate protection at 2-OH. Neighbouring group participation due to the forma-

tion of the acyloxonium ion – formation of 1,2-trans glycosides. ................................................ 8

Scheme 1.5 - Benzyl ether protection at 2-OH and non-neighbouring group participation. ........ 8

Scheme 2.1 – Synthetic strategy followed for the synthesis of the glycosyl donor 11. Reagents

and conditions: i) allylic alcohol, camphorsulfonic acid, Δ, overnight, 94%; ii) benzaldehyde

dimethyl acetal, camphorsulfonic acid, THF, Δ, 4 hours: 30 minutes, 59 %; iii) dibutyltin oxide,

methanol, Δ, 3 hours and iv) iodomethane, DMF, 50 ºC, overnight, 2 steps: 80 %; v) acetic

anhydride, DMAP, 0ºC → rt, pyridine 2 hours; 91 %; vi), sodium cyanoborohydride, hydrogen

chloride in diethyl ether 1 M, THF, 0ºC, 81 %; vii) acetic anhydride, DMAP, pyridine,

0ºC → rt, 1 hour: 30 minutes; 88 %; viii) palladium (II) chloride, methanol, rt, 2 hours; 75 %;

ix) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 10 minutes, 76 %. ............................ 14

Scheme 2.2 - Synthesis of Allyl (α/β)-D-mannopyranoside 3 with i) allylic alcohol, camphor-

sulfonic acid, Δ, overnight, 94%. ................................................................................................ 14

Scheme 2.3 - Mechanism for the synthesis of Allyl (α/β)-D-mannopyranoside 3. .................... 15

Scheme 2.4 - Synthesis of Allyl 4,6-O-Benzylidene-(α/β)-D-mannopyranoside 4 with ii) benzal-

dehyde dimethyl acetal, camphorsulfonic acid, THF, Δ, 4 hours: 30 minutes, 59 %. ............... 15

Scheme 2.5 - Mechanism for the synthesis of Allyl 4,6-O-benzylidene-(α/β)-D-mannopyrano-

side 4. .......................................................................................................................................... 16

Scheme 2.6 - Synthesis of Allyl 4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranoside 6

with iii) dibutyltin oxide, methanol, Δ, 3 hours and iv) iodomethane, DMF, 50 ºC, overnight; 2

steps: 80 %. ................................................................................................................................. 16

Scheme 2.7 – Synthesis of Allyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-

mannopyranoside 7 with v) acetic anhydride, DMAP, 0ºC → rt, pyridine 2 hours; 91 %. ........ 17

Scheme 2.8 - a) Mechanism for the formation of the acylpyridinium cation; b) Mechanism for

the synthesis of Allyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranoside 7.

..................................................................................................................................................... 17

Scheme 2.9 – Synthesis of Allyl 2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside

8 with vi) sodium cyanoborohydride, hydrogen chloride in diethyl ether 1 M, THF, 0ºC,

81 %. ........................................................................................................................................... 18

Scheme 2.10 – Mechanism for the synthesis of Allyl 2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-

-D-mannopyranoside 8. ............................................................................................................... 19

Scheme 2.11 – Synthesis of Allyl 2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyra-

noside 9 with vii) acetic anhydride, DMAP, pyridine, 0ºC → rt, 1 hour: 30 minutes; 88 %. ..... 19

XIV

Scheme 2.12 - Synthesis of 2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose

10 with viii) palladium (II) chloride, methanol, rt, 2 hours; 75 %. ............................................. 20

Scheme 2.13 - Mechanism for the synthesis of Allyl 2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-

(α/β)-D-mannopyranose 10. ........................................................................................................ 20

Scheme 2.14 – Synthesis of (2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-

mannopyranosyl)-trichloroacetimidate 11 with ix) DBU and trichloroacetonitrile,

dichloromethane, 0ºC, 10 minutes, 76 %. ................................................................................... 21

Scheme 2.15 – Synthetic strategy followed for the synthesis of the glycosyl acceptor 16.

Reagents and conditions: i) benzaldehyde dimethyl acetal, camphorsulfonic acid, THF, Δ,

overnight, 50 %; ii) dibutyltin oxide, methanol, Δ, overnight and iii) iodomethane, DMF, 65 ºC,

overnight, 2 steps: 50%; iv) acetic anhydride, DMAP, 0ºC → rt, pyridine 2 hours; 97%; v),

sodium cyanoborohydride, hydrogen chloride in diethyl ether 1 M, THF, 0ºC, 100 %.............. 22

Scheme 2.16 – Synthesis of Methyl 4,6-O-benzylidene-α-D-mannopyranoside 12 with i)

benzaldehyde dimethyl acetal, camphorsulfonic acid, THF, Δ, overnight, 50 %. ...................... 22

Scheme 2.17 – Synthesis of Methyl 4,6-O-benzylidene-3-O-methyl-α-D-mannopyranoside 14

with ii) dibutyltin oxide, methanol, Δ, overnight and iii) iodomethane, DMF, 65 ºC, overnight; 2

steps: 50%. .................................................................................................................................. 23

Scheme 2.18 – Synthesis of Methyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-α-D-

mannopyranoside 15 with iv) acetic anhydride, DMAP, 0ºC → rt, pyridine 2 hours; 97%. ...... 23

Scheme 2.19 – Synthesis of Methyl 2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside

16 with v), sodium cyanoborohydride, hydrogen chloride in diethyl ether 1 M, THF, 0ºC, 100

%. ................................................................................................................................................ 24

Scheme 2.20 – Synthetic route followed for the synthesis of the disaccharide 1. Reagents and

conditions: i) TMSOTf, dichloromethane, -20 ºC, 30 minutes, 69 %; ii) sodium methoxide,

methanol, rt, 2 hours: 30 minutes, 98%; iii) H2/Pd/C 10%, ethyl acetate /ethanol 1:1, 50 psi,

overnight, 100 %. ........................................................................................................................ 24

Scheme 2.21 – Synthesis of Methyl (2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-α-D-

mannopyranosyl)-(1→4)-2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 17 with i)

TMSOTf, dichloromethane, -20 ºC, 30 minutes, 69 %. .............................................................. 25

Scheme 2.22 - Mechanism for the glycosylation reaction and synthesis of 17. ......................... 26

Scheme 2.23 - Synthesis of Methyl (6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-6-

O-benzyl-3-O-methyl-α-D-mannopyranoside 18 with ii) sodium methoxide, methanol, rt, 2

hours: 30 minutes, 98%. .............................................................................................................. 27

Scheme 2.24 – Synthesis of Methyl (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-

D-mannopyranoside 1 with iii) H2/Pd/C 10%, ethyl acetate /ethanol 1:1, 50 psi, overnight, 100

%. ................................................................................................................................................ 27

Scheme 2.25 – Synthetic route followed for the synthesis of the disaccharide 2. Reagents and

conditions: i) TMSOTf, dichloromethane, -20 ºC, 30 minutes, 77 %; ii) palladium (II) chloride,

methanol, rt, 2 hours; 80%; iii) sodium methoxide, methanol, rt, 6 hours: 30 minutes, 78%; iv)

H2/Pd/C 10%, ethyl acetate /ethanol 5:1, 50 psi, 7 hours, 98 %. ................................................ 28

Scheme 2.26 – Synthesis of Allyl (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-α-D-

mannopyranosyl)-(1→4)-2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 19 with

i) TMSOTf, dichloromethane, -20 ºC, 30 minutes, 77 %. ........................................................... 28

Scheme 2.27 – Synthesis of (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-

(1→4)-2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 20 with ii) palladium (II)

chloride, methanol, rt, 2 hours; 80%. .......................................................................................... 29

XV

Scheme 2.28 – Synthesis of (6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-6-O-

benzyl-3-O-methyl-(α/β)-D-mannopyranoside 21 with iii) sodium methoxide, methanol, rt, 6

hours: 30 minutes, 78%. .............................................................................................................. 29

Scheme 2.29 - Synthesis of (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-(α/β)-D-

mannopyranoside 2 with iv) H2/Pd/C 10%, ethyl acetate /ethanol 5:1, 50 psi, 7 hours, 98 %. ... 30

Scheme 2.30 – Synthetic route followed for the synthesis of the disaccharide glycosyl acceptor

24. Reagents and conditions: a) TMSOTf, dichloromethane, -20 ºC, 30 minutes. ..................... 31

Scheme 2.31 – Synthetic route proposed for the synthesis of the glycosyl donor 32. Reagents

and conditions: i) DIPEA, TBDMSOTf, dichloromethane, 0 ºC, 20 minutes, 89%. .................. 32

Scheme 2.32 - Synthesis of Allyl 2-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-

methyl-(α/β)-D-mannopyranoside 25 with i) DIPEA, TBDMSOTf, dichloromethane, 0 ºC, 20

minutes, 89%. .............................................................................................................................. 32

Scheme 2.33 – Attempted synthesis of 2-O-Acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-

O-methyl-(α/β)-D-mannopyranose 26 with ii) bis(dibenzylideneacetone)palladium (0),

1,4-Bis(diphenylphosphino)butane, THF, rt, 15 minutes and iii) 1,3-dimethylbarbituric acid,

THF, 60 ºC, overnight. ................................................................................................................ 33


O-methyl-(α/β)-D-mannopyranose 26 with iv) sodium borohydride, iodine, THF, 0 ºC, 3 hours

and 20 minutes. ........................................................................................................................... 33

Scheme 2.35 - Mechanism for the synthesis of 2-O-Acetyl-6-O-benzyl-4-O-tert-

butyldimethylsilyl-3-O-methyl-(α/β)-D-mannopyranose 26 using the described reaction

conditions.[16]

............................................................................................................................... 34


O-methyl-(α/β)-D-mannopyranose 26 with v) t-BuOK, DMF, 60 ºC, 1 hour. .......................... 34


O-methyl-(α/β)-D-mannopyranose 26 with vi) acetic acid/H2O (90 % v/v), sodium acetate,

palladium (II) chloride, ethyl acetate, rt, overnight. .................................................................... 35

Scheme 2.38 – Mechanism for the Wacker oxidation. ............................................................... 38

Scheme 2.39 - Attempted synthesis of 2-O-Acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-

methyl-(α/β)-D-mannopyranose 26 with vii) (dimethyl sulfide)trihydroboron, THF, 0 ºC, 20

minutes. ....................................................................................................................................... 39

Scheme 2.40 – Alternative synthetic route proposed for the synthesis of the glycosyl donor 32.

Reagents and conditions: i) acetic acid/H2O (90 % v/v), sodium acetate, palladium (II) chloride,

ethyl acetate, rt, overnight 73%; ii) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 10

minutes, 36 %; iii) DIPEA, TBDMSOTf, dichloromethane, 0 ºC, 20 minutes, 74 %. ............... 40

Scheme 2.41 – Synthesis of 2-O-Acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose 29

with i) acetic acid/H2O (90 % v/v), sodium acetate, palladium (II) chloride, ethyl acetate, rt,

overnight 73%. ............................................................................................................................ 40

Scheme 2.42 – Synthesis of (2-O-Acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-mannopyranosyl)-

trichloroacetimidate 30 with ii) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 10

minutes, 36 %. ............................................................................................................................. 40

Scheme 2.43 – Synthesis of (2-O-0Acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-

1-O-α-D-mannopyranosyl)-trichloroacetimidate 32 with iii) DIPEA, TBDMSOTf,

dichloromethane, 0 ºC, 20 minutes, 74 %. .................................................................................. 41

Scheme 2.44 - Synthesis of Methyl (2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-

(1→4)-2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 24 with iv) TMSOTf,

dichloromethane, -20 ºC, 30 minutes, 18 %. ............................................................................... 42

XVI

Scheme 2.45 – Synthetic route proposed for the synthesis of the glycosyl donor 34. Reagents

and conditions: i) acetic acid/H2O (90 % v/v), sodium acetate, palladium (II) chloride, ethyl

acetate, rt, 5 hours, 78%; ii) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 3 hours, 10%.

..................................................................................................................................................... 42

Scheme 2.46 – Synthesis of 2-O-Acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-

mannopyranose 33 with i) acetic acid/H2O (90 % v/v), sodium acetate, palladium (II) chloride,

ethyl acetate, rt, 5 hours, 78%. .................................................................................................... 43

Scheme 2.47 – (2-O-Acetyl-4,6-O-benzylidene-3-O-methyl-1-O-α-D-mannopyranosyl)-tri-

chloroacetimidate 34 with ii) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 3 hours,

10 %. ........................................................................................................................................... 43

Scheme 2.48 – Alternative synthetic route followed for the synthesis of the glycosyl donor

32. ................................................................................................................................................ 44

Scheme 2.49 – Attempted synthesis of Methyl 3-O-methyl-α-D-mannopyranoside 35 with i)

dibutyltin oxide, toluene, Δ, 3 hours; ii) iodomethane, TBAI, toluene, 70 ºC, 72 hours. ........... 44

Scheme 2.50 – Synthesis of Methyl 3-O-methyl-α-D-mannopyranoside 35 with iii) dibutyltin

oxide, methanol, Δ, overnight; iv) iodomethane, DMF, 65 ºC, overnight. ................................. 45

Scheme 2.51 – Alternative route proposed for the synthesis of the glycosyl donor 32. Reagents

and conditions: i) TrCl, pyridine, rt, 24 hours; ii) TrCl, DMAP, pyridine rt, overnight; 2 steps:

100 %; iii) dibutyltin oxide, methanol, Δ, overnight; iv) iodomethane, DMF, 65 ºC, overnight; 2

steps : 68 %; v) acetic anhydride/acetic acid/sulfuric acid 105:45:1, v/v/v, rt, overnight. .......... 46

Scheme 2.52 – Synthesis of Methyl 6-O-trityl-α-D-mannopyranoside 36 with i) TrCl, pyridine,

rt, 24 hours; ii) TrCl, DMAP, pyridine rt, overnight; 2 steps: 100 %. ........................................ 46

Scheme 2.53 – Synthesis of Methyl 3-O-methyl-6-O-trityl-α-D-mannopyranoside 37 with iii)

dibutyltin oxide, methanol, Δ, overnight; iv) iodomethane, DMF, 65 ºC, overnight; 2 steps:

68%. ........................................................................................................................................... 47

Scheme 2.54 – Synthesis of Methyl 1,2,4,6-tetra-O-acetyl-3-O-methyl-(α/β)-D-mannopyranose

38 with v) acetic anhydride/acetic acid/sulfuric acid 105:45:1, v/v/v, rt, overnight. .................. 47

Scheme 2.55 – Synthesis of the disaccharide glycosyl donor 23. Reagents and conditions: a)

DBU and trichloroacetonitrile, dichloromethane, 0ºC, 10 minutes. ............................................ 48

XVII

List of Figures

Figure 1.1 – The structure of the three MMP cellular precursors. ............................................... 3

Figure 1.2 - The structure of mycobacterial MMPs. .................................................................... 4

Figure 1.3 - Two different glycosylation products, the α- and the β-O-glycoside. ...................... 6

Figure 2.1 - The structure of Methyl (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-

D-mannopyranoside 1 and (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-(α/β)-D-

mannopyranose 2. ....................................................................................................................... 13

Figure 2.2 - Structure of Allyl 4,6-O-benzylidene-2,3-O-dibutylstannylene-(α/β)-D-

mannopyranoside 5. .................................................................................................................... 16

Figure 2.3 – The structure of Methyl (3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-

D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-

mannopyranoside 22. .................................................................................................................. 31

Figure 2.4 – The structure of the glycosyl donor (2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-α-D-

mannopyranosyl-(1→4)-2-O-Acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-mannopyranosyl)-

trichloroacetimidate 23 and the glycosyl acceptor Methyl (2-O-Acetyl-6-O-benzyl-3-O-methyl-

α-D-mannopyranosyl)-(1→4)-2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 24. 31

Figure 2.5 –1H-NMR spectrum, corresponding to the mixture of two pairs of doublets (between

δ 4.65 and 4.50 ppm) and two doublets (at δ 4.24 ppm and 4.13 ppm). ..................................... 36

Figure 2.6 – The structure of 1,2-di-O-Acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-

methyl-α-D-mannopyranose 27. ................................................................................................. 36

Figure 2.7 – 1H-NMR spectra overlay from the two obtained compounds (between δ 6.2 and 4.0

ppm). The green spectrum is from compound 27 and the red one is from compound 28. .......... 37

Figure 2.8 – 13

C-APT spectra overlay from the two obtained compounds (between δ 220.0 and -

20.0 ppm). The green spectrum is from compound 27 and the red one is from compound 28. .. 37

Figure 2.9 – Two possible products, which result from the Wacker oxidation of the allyl group.

..................................................................................................................................................... 38

Figure 2.10 – The structure of (2-O-Acetyl-6-O-benzyl-3-O-methyl-1,4-O-α-D-mannopyra-

nosyl)-di-trichloroacetimidate 31. ............................................................................................... 41

XVIII

XIX

Abbreviations and Symbols

ABdd – AB doublet of doublets

Ac – Acetyl

Ar - Aromatics

ATR-FTIR – Attenuated Total Reflectance-Fourier Transform Infra-red Spectroscopy

br – Broad

Bn – Benzyl

Bu - Butyl

COSY – Correlation Spectroscopy

13C-NMR - Carbon-13 nuclear magnetic resonance

d – Doublet

DBU- 1,8-Diazabicycloundec-7-ene

dd – Doublet of doublets

ddd - Doublet of doublet of doublets

DIPEA – N,N-Diisopropylethylamine

DMAP - 4-Dimethylaminopyridine

DMF – Dimethylformamide

HMQC - Heteronuclear Multiple-Quantum Correlation

1H-NMR – Proton nuclear magnetic resonance

IR – Infra-Red

J – Coupling constant

Me – methyl

m – Multiplet

Nu - Nucleophile

Ph – Phenyl

rt – Room temperature

s – Singlet

TBDMSOTf - tert-Butyldimethylsilyl triflate

XX

THF – Tetrahydrofuran

TLC – Thin Layer Chromatography

TMSOTf - Trimethylsilyl trifluoromethanesulfonate

t – Triplet

TrCl – Trityl chloride

t-BuOK- Potassium tert-butoxide

UV – Ultraviolet

δ – Chemical shift

Δ - Reflux

Mannose carbon numeration:

Mannose disaccharide monomer identification:

1

CHAPTER 1

INTRODUCTION

2

3

1. Introduction Organic synthesis is often connected with several biological fields because it allows the

synthesis of several compounds, which can become potential drugs, or even assist the

understanding of some cellular metabolic processes. Carbohydrates are present on most cells as

glycoproteins, glycopeptides or polysaccharides, and they have important functions, such as

cell-wall receptors during many biological processes. The objective of this work is the synthesis

of three saccharides, which are cellular precursors for the biosynthesis of rare mycobacterial

polysaccharides, 3-O-methyl-mannose polysaccharides (MMPs). The structures of these

compounds are shown in Figure 1.1.

Figure 1.1 – The structure of the three MMP cellular precursors.

1.1 Location and biological function of MMPs

Mycobacterium is a genus of Actinobacteria, which includes pathogens known to cause

serious diseases, including tuberculosis, leprosy, pulmonary disease resembling tuberculosis or

lymphadenitis. Mycobacteria in general synthesize some types of cytoplasmic carbohydrates,

polymethylpolysaccharides (PMPs), which play important roles in metabolism regulation, like

for example lipid metabolism. Some of these microorganisms produce a class of PMPs, 3-O-

-methylmannose polysaccharides (MMPs). These are composed of 10-13 α-(1→4)-linked 3-O-

-methyl-D-mannoses.[1]

The nonreducing end of these compounds is terminated by a single

α-linked unmethylated D-mannose and the reducing end by an α-methyl aglycon.[1]

The

structures of MMPs are shown in Figure 1.2.

4

Figure 1.2 - The structure of mycobacterial MMPs.

In this work, these molecules have some particular interest, because of their important

biological functions in mycobacteria. MMPs have been detected only in nontuberculous

mycobacteria (NTM), like for example in Mycobacterium smegmatis.[1]

These are the

mycobacteria which can cause pulmonary disease resembling tuberculosis or lymphadenitis.

One of the intracellular functions of MMPs is the formation of a stable 1:1 complex with long-

chain fatty acids and acyl coenzyme A (acyl-CoA), because of the helically coiled conformation

of this polysaccharide, which enables it to include the lipid in its interior in a specific

orientation.[1]

The 3-O methylation has an important role in the stabilization of the helical

conformation of this polysaccharide and also enhances the direct interaction with lipids and

acyl-CoA, because methyl groups are hydrophobic.[1]

The formation of these stable complexes,

allows MMPs to be used as intracellular lipid carriers, regulators of the fatty acid synthesis,

because they can activate or inhibit the fatty acid synthetase complex (FAS-I), and regulators of

the length of the fatty acid chain, due to the fact that they can also facilitate the release of the

neo-synthesized fatty-acid chains from the FAS-I, terminating their elongation.[1][2]

Recent studies revealed that these molecules synthesized by these species of

mycobacteria have a significant role as a potential antigen or target for new vaccines and drugs

used in tuberculosis disease treatment and diagnosis.[1]

Besides that, the synthesis of saccharide

precursors that are MMP intermediates allows the study of their biosynthetic pathways on these

microorganisms, because these molecules are going to be important in enzyme identification,

characterization and functional validation. After studying this pathway it will be confirmed if

the inhibition of the synthesis of MMP from M. smegmatis will critically affect the survival of

these microorganisms.

1.2 Chemical synthesis of mannose oligosaccharides

For the synthesis of any poly- or oligosaccharide, it is initially necessary to create the

bond between two precursors (monosaccharides) in a process that is called glycosylation

reaction (Scheme 1.1). After the formation of this disaccharide the compound may react again

5

with other saccharides, in the same process, making an oligosaccharide or a polysaccharide,

depending on the number of monomers that the final molecule has.

At the end of the nineteenth century, Emil Fischer and other chemists showed that the

formation of this glycosidic bond could be done by a chemical process. However, these

scientists also verified the complexity of the glycosylation reaction. After these first attempts,

there was a huge development in the study of this chemical process, but only in the last twenty

years the scientific community had reached a major advance of the methods used for this

reaction.[3]

The development of new strategies has not only allowed the access to novel types of

glycosidic linkages but also led to the discovery of efficient strategies for the synthesis of

several oligosaccharides and polysaccharides.

This important work allowed to understand that some crucial factors must be

considered. The two precursors for this reaction must have special characteristics so that this

reaction can actually happen. One of these precursors, the glycosyl donor, must have in its

anomeric carbon a leaving group (LG). The second one, the glycosyl acceptor, must possess a

free hydroxyl group so that he can react with the anomeric carbon of the donor, just like a

nucleophile (Scheme 1.1).

Scheme 1.1 - The chemical glycosylation reaction between two monosaccharides.

This initial process will enable the growth of the oligosaccharide (or the

polysaccharide).[4]

There are some limitations in this kind of reaction. The glycosylation has to

be, for example, carried out in anhydrous conditions, because of the formation of by-products

that result from the hydrolysis of the glycosyl donor in the presence of water (Scheme 1.2).

Scheme 1.2 – Hydrolysis of the glycosyl donor.

6

Besides the necessity of anhydrous conditions during the reaction (assured by adding

molecular sieves to the reaction, performing it under an inert environment and using dry

solvents) it is important to consider other factors such as:

- Regioselectivity, because only one hydroxyl group of the acceptor precursor has to

react with the anomeric carbon of the donor;

- Stereoselectivity, because the product that is formed must be predominantly α or β;

- Efficiency, because alcohols are not good nucleophiles, so, many strategies are

taken to improve the yield of the reaction, like for example a good leaving group at

the donor.[4]

1.2.1 Mechanism

1.2.1.1 SN1 reaction

Chemical glycosylation is a substitution reaction, because the acceptor, which has a free

hydroxyl group, reacts, as a nucleophile, with the anomeric carbon of the donor, affording a

glycosidic bond.[4]

This reaction follows very often a unimolecular mechanism (SN1),[4]

mostly

because sugar acceptors are very weak nucleophiles and the fact that the oxygen linked to the

anomeric carbon has two non-bonding electron pairs that facilitate the departure of the LG,

which is a very good leaving group [4]

:

Scheme 1.3 – Departure of the leaving group and formation of the oxonium ion.

This interaction can be described as a n → σ* donation. After the formation of this

oxonium ion, the nucleophile can react with this intermediate. However, this nucleophilic attack

can be made in two ways, giving two different products, β and α:

Figure 1.3 - Two different glycosylation products, the α- and the β-O-glycoside.

α β

7

The formation of this oxonium ion, and the use of this very good LG is crucial for the

efficiency of the glycosylation reaction, because, in most cases, it allows a unimolecular

substitution reaction (SN1), so, the anomeric carbon becomes more electrodeficient and more

capable to be attacked even by a weak nucleophile, like the acceptor.[3]

So, leaving groups are

very important in the chemical glycosylation because most donors are too stable to undergo

spontaneous glycosylation. Depending on the kind of glycosyl donor and final product, there are

several types of leaving groups, such as halides, trichloroacetimidates, thioglycosides, acetates,

phosphites, etc.[4]

However, most leaving groups first have to be activated, before their

departure from the molecule, during the glycosylation reaction. Promoters (activators) are used

to form an activated species with the LG and that will eventually lead to its departure.[5]

Other factors can increase the stereoselectivity of the product, such as the solvent, the

protecting groups at 2-OH and other positions.

1.2.1.2 Protecting groups

In carbohydrate chemistry protecting groups like allyl ether, silyl ethers (TBDMS or

TBDPS), acetals, benzyl ethers or the acetyl group, are used for the protection of sugar

hydroxyls, allowing a regioselective reaction, since only one hydroxyl group of the acceptor is

free to react with the anomeric carbon of the mannosyl donor. Besides that, one of the powerful

strategies used to positively influence the stereoselectivity outcome of the reaction, is also the

use of those protecting groups, like an ester or ether, at the neighbouring group (C-2 carbon).

Acetyl

Acetyl is a very common protecting group used in carbohydrate chemistry. The

existence of an acetyl protecting group at the 2-OH allows the nucleophilic attack on only one

side of the molecule, because of the formation of an intermediate, the acyloxonium ion, that

results from the attack of the acetate carbonyl oxygen to the anomeric carbon (neighbouring

group participation). This cyclic oxonium ion can be opened by a bimolecular nucleophilic

substitution (SN2) reaction by the reacting nucleophile.[3,4]

The new bond formed is also trans

compared with the 2-OH (Scheme 1.4). With the acetyl protection at 2-OH, the 1,2-trans

stereoselectivity is strongly favored.

8

Scheme 1.4 – Acetyl protection at 2-OH. Neighbouring group participation due to the formation of the

acyloxonium ion – formation of 1,2-trans glycosides.

Benzyl ether

Benzyl ether is also used as a protecting group at 2-OH, but since there is not any

neighbouring group participation, a mixture of anomers, which result from the nucleophilic

attack on both sides of the molecule, are formed (Scheme 1.5). However, there is a slight

stereochemical outcome for glycosyl donors with this nonparticipating group at 2-OH, due to

the existence of an anomeric effect, which favors the α-product.[3]

Even so, the fact that the

glycosylation is irreversible, makes the role of the anomeric effect diminished.[3]

Because of

that, benzyl ether is often used as a neighbouring group in the chemical formation of

β-mannosides, for example, but in this case there are other factors which influence the

stereochemistry of the final product, like for example the solvent.[3]

Scheme 1.5 - Benzyl ether protection at 2-OH and neighbouring group non-participation.

Comparing to the effect of acetyl protection at 2-OH, the appearance of stereoselectivity

is obviously less favored, because of the absence of a participating group.

α > β

α > β

9

1.2.1.3 Solvent effect

In a glycosylation reaction the solvent is another important factor which influences the

stereoselectivity at the anomeric center of the final molecule. The use of polar solvents increases

the formation rate of β-glycosides. Non-polar solvents, such as dichloromethane or toluene, are

used in the synthesis of α-glycosides.[4]

This work will also highlight the importance of chemical glycosylation, which in this

case can be an important alternative to enzymatic glycosylation, since the first one can solve

many problems which enzymatic glycosylation cannot.

10

11

CHAPTER 2

RESULTS AND DISCUSSION

12

13

2. Results and discussion

As it was said before, the objective of this work is an efficient synthesis of three rare

cellular precursors, which are saccharides used in the biosynthesis of rare mycobacterial

polysaccharides - MMPs. Since the commercially available compounds are monosacharides, in

order to obtain the desired products with good yields, some crucial factors on the glycosylation

reaction must be considered, such as its regioselectivity, efficiency and stereoselectivity.

Besides that, the desired compounds have to be methylated in specific positions, so the strategy

of the synthesis also has to include good regioselective methylation steps.

2.1 Disaccharide synthesis

Two of the proposed objectives was the synthesis of the disaccharides methyl (3-O-

methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-D-mannopyranoside and (3-O-methyl-α-D-

mannopyranosyl)-(1→4)-3-O-methyl-(α/β)-D-mannopyranose (Figure 2.1).

Figure 2.1 - The structure of methyl (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-D-

mannopyranoside 1 and (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-(α/β)-D-mannopyranose

2.

In order to fulfill the conditions mentioned above, the mannose precursors need to be

prepared for the glycosylation reaction. To accomplish that, the synthetic strategies for the

preparation of the mannosyl donor and acceptor were drawn.

2.1.1 Monosaccharide glycosyl donor synthesis

D-mannose was used as starting material for the formation of the glycosyl donor.

During this synthesis, the configuration of the anomeric carbon is not important. Only after the

formation of the disaccharide, the formed glycosidic bond must have the right anomeric

configuration.

An efficient synthetic pathway for the synthesis of a 3-O-methyl mannose glycosyl

donor has been reported.[2]

Benzyl ether was used as protecting group at 2-OH. However, as it

was said before, an acetyl protecting group at 2-OH offers better stereochemical results on the

glycosylation reaction. Besides that, this pathway includes an efficient 3-O-methylation step,

14

which facilitates one of the challenges mentioned above. In conclusion, a synthetic strategy

based on the reported work [2]

was drawn with some additional changes.

The proposed synthetic route is show below in Scheme 2.1.

Scheme 2.1 – Synthetic strategy followed for the synthesis of the glycosyl donor 11. Reagents and

conditions: i) allylic alcohol, camphorsulfonic acid, Δ, overnight, 94%; ii) benzaldehyde dimethyl acetal,

camphorsulfonic acid, THF, Δ, 4 hours: 30 minutes, 59 %; iii) dibutyltin oxide, methanol, Δ, 3 hours and

iv) iodomethane, DMF, 50 ºC, overnight, 2 steps: 80 %; v) acetic anhydride, DMAP, 0ºC → rt, pyridine 2

hours; 91 %; vi), sodium cyanoborohydride, hydrogen chloride in diethyl ether 1 M, THF, 0ºC, 81 %;

vii) acetic anhydride, DMAP, pyridine, 0ºC → rt, 1 hour: 30 minutes; 88 %; viii) palladium (II) chloride,

methanol, rt, 2 hours; 75 %; ix) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 10 minutes, 76 %.

2.1.1.1 Allyl (α/β)-D-mannopyranoside 3 synthesis

Scheme 2.2 - Synthesis of allyl (α/β)-D-mannopyranoside 3 with i) allylic alcohol, camphorsulfonic acid,

Δ, overnight, 94%.

Liao et al synthetic route[2]

uses allyl α-D-mannopyranoside as starting material, which

is an expensive reagent. However, since a reported procedure [6]

of D-mannose 1-O-allylation

using allylic alcohol (as reagent and solvent) and camphorsulfonic acid as acid catalyst, under

reflux, gives very good yields, it is not necessary to use allyl α-D-mannopyranoside as starting

material. The reason why this allylation is regioselective is because camphorsulfonic acid, as a

source of protons, catalyses the formation of the oxonium ion. After that, the allylic alcohol will

attack the anomeric carbon on both sides of the molecule (Scheme 2.3).

15

Scheme 2.3 - Mechanism for the synthesis of allyl (α/β)-D-mannopyranoside 3.

This reaction follows a unimolecular mechanism (SN1), due to the fact that the acid

catalyst and the oxygen linked to the anomeric carbon facilitate the departure of the leaving

group. The resulting product is not only the allyl α-D-mannopyranoside, due to the nucleophilic

attack on both sides of the molecule, which also leads to the formation of the allyl β-D-

mannopyranoside. As it was said before, the configuration of the anomeric carbon will not be

important during the synthesis of the glycosyl donor. The allyl ether as protecting group has

been frequently used in carbohydrate research, mostly because it has great advantages in

comparison with other protecting groups, such as the fact that it is a very stable group.

Unfortunately the same advantages can sometimes bring disadvantages, as it will be seen further

in this work. Interpretation of the 1H-NMR spectrum revealed that the obtained product was the

pretended compound with a yield of 94 %.

2.1.1.2 Allyl 4,6-O-benzylidene-(α/β)-D-mannopyranoside 4 synthesis

Scheme 2.4 - Synthesis of allyl 4,6-O-benzylidene-(α/β)-D-mannopyranoside 4 with ii) benzaldehyde

dimethyl acetal, camphorsulfonic acid, THF, Δ, 4 hours: 30 minutes, 59 %.

The synthesis of 4 consists in a regioselective formation of a 4,6-O benzylidene acetal,

catalyzed by camphorsulfonic acid, using benzaldehyde dimethyl acetal as reagent and THF as

solvent, all stirred under reflux. The reason why this cyclic diol protection is 4,6-O

regioselective, is due to the thermodynamic control on the reaction, which favors the formation

of a six-membered benzylidene acetal ring, a very stable product.[7]

The phenyl group is

oriented in an equatorial orientation.

In this acid-catalysed acetalation, camphorsulfonic acid is used as the catalyst and it

activates benzaldehyde dimethyl acetal.[8]

The protonated methoxy group of the reagent can be

displaced by the sugar primary hydroxyl group and gives a mixed acetal. The protonation of the

second methoxy group and its further displacement gives the formation of an oxocarbenium ion.

The second hydroxyl group from the molecule reacts with this ion, and gives the protonated

16

acetal, which after deprotonation (the catalyst is regenerated) results in the cyclic acetal

(Scheme 2.5).

Scheme 2.5 - Mechanism for the synthesis of allyl 4,6-O-benzylidene-(α/β)-D-mannopyranoside 4.

Interpretation of the 1H-NMR spectrum revealed that the obtained product was the

pretended compound with a yield of 59%. The use of benzylidene acetal as a protecting group in

this synthetic strategy is important, because it has some advantages, such as the fact that it can

be introduced in the molecule under acidic conditions, it protects the compound in the 4 and 6

positions, even with the rest of the positions available to be protected, and it can be

regioselectively opened, as it will be seen further in this work.

2.1.1.3 Allyl 4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranoside 6 synthesis

Scheme 2.6 - Synthesis of allyl 4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranoside 6 with iii)

dibutyltin oxide, methanol, Δ, 3 hours and iv) iodomethane, DMF, 50 ºC, overnight;

2 steps: 80 %.

The synthesis of 6 consists in a regioselective 3-O methylation. A two step described

procedure[9]

was applied to 4, using more quantity of dibutyltin oxide. The reason why this

reaction is 3-O regioselective is because of the dibutyltin oxide, which reacts with the

mannopyranoside, under reflux in methanol, and forms the cyclic 2,3-O-di-butylstannylene

intermediate 5, shown in Figure 2.2.

Figure 2.2 - Structure of allyl 4,6-O-benzylidene-2,3-O-dibutylstannylene-(α/β)-D-mannopyranoside 5.

17

After the formation of the intermediate, iodomethane is added, in DMF at 50 ºC. The

reagent is going to selectively methylate the equatorial hydroxyl group.[10]

Despite some

hypothesis,[11]

it is not clear why reactions using organotin derivatives are regioselective, such

as the mechanism. Interpretation of the 1H-NMR spectrum revealed that the obtained product

was the pretended compound with a yield of 80 %. Hsu et al[9]

described procedure was efficient

and one of the challenges of the synthesis was achieved.

The reason why the synthetic route for the synthesis of the glycosyl donor did not start

with the methylation of 3, as it was reported by Liao et al[2]

, is going to be explained further in

this work.

2.1.1.4 Allyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranoside 7

synthesis

Scheme 2.7– Synthesis of allyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranoside 7

with v) acetic anhydride, DMAP, 0ºC → rt, pyridine 2 hours; 91 %.

The synthesis of 7 consists in an acetyl protection of 2-OH, using acetic anhydride as

reagent, DMAP as catalyst, and pyridine as solvent.

DMAP first reacts with acetic anhydride, and forms an acylpyridinium cation (Scheme

2.8a). The free hydroxyl from the sugar then reacts with the acylated catalyst to form the ester

product[12]

(Scheme 2.8b). Pyridine will neutralize the acetic acid formed.

Scheme 2.8 - a) Mechanism for the formation of the acylpyridinium cation; b) Mechanism for the

synthesis of allyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranoside 7.

18

This important step is going to influence, as it was said before, the stereoselectivity of

the glycosylation reaction. Fortunately, the acetyl group can be introduced and removed in the

molecule very easily, which justifies the fact that it is one of the most important protecting

groups used in carbohydrate chemistry. It was important to proceed to this acetylation step with

the molecule containing only one free hydroxyl, because it is not a regioselective reaction.

Interpretation of the 1H-NMR spectrum revealed that the obtained product was the pretended

compound with a yield of 88 %.

2.1.1.5 Allyl 2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 8

synthesis

Scheme 2.9 – Synthesis of allyl 2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 8 with vi),

sodium cyanoborohydride, hydrogen chloride in diethyl ether 1 M, THF, 0ºC, 81 %.

Benzylidene acetal can be either removed from the molecule, by for example acidic

hydrolysis, or opened regioselectively using different methods.[13]

The synthesis of 8 consists in

a regioselective reductive opening of the benzylidene acetal from 7. A described procedure [2]

was applied to the compound, with some changes. 7 and sodium cyanoborohydride were

dissolved in THF, and hydrogen chloride in diethyl ether was added portionwise, at 0ºC, until

the reaction was finished.

Hydrogen chloride acts in this reaction as a Brønsted acid and reacts with cyanoboro-

hydride, to give H2BCN and H2. The formed borane, activated by the acid, is electrophilic

enough to form an initial complex with the most electron rich oxygen of the acetal (6-O). The

reason why the solution was added portionwise is because the acid must not be added in excess,

as it can provoke the degradation of the molecule. This reaction proceeds through an

oxocarbenium ion, which is reduced by the borane to give the pretended compound, following

the proposed mechanism[13]

(Scheme 2.10).

19

Scheme 2.10 – Mechanism for the synthesis of allyl 2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-

mannopyranoside 8.

The reason why this reaction has to use a large excess of sodium cianoborohydride (12

equivalents) is unknown. An experiment using less quantity of this compound (6 equivalents)

was performed and afforded the expected product but with a lower yield (60 %). However,

using the other conditions (12 equivalents), interpretation of the 1H-NMR spectrum revealed

that the obtained product was the pretended compound with a yield of 81 %.

2.1.1.6 Allyl 2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 9

synthesis

Scheme 2.11 – Synthesis of allyl 2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 9

with vii) acetic anhydride, DMAP, pyridine, 0ºC → rt, 1 hour: 30 minutes; 88 %.

The synthesis of 9 consists in an acetylation of the 4-OH, using acetic anhydride,

DMAP and pyridine. Interpretation of the 1H-NMR spectrum revealed that the obtained product

was the pretended compound with a yield of 88 %. It is very important to have all the hydroxyls

protected, and the reason will be seen in the next steps of the synthetic route.

20

2.1.1.7 2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose 10

synthesis

Scheme 2.12 - Synthesis of 2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose 10 with

viii) palladium (II) chloride, methanol, rt, 2 hours; 75 %.

The allyl ether has been frequently used as protecting group in carbohydrate research. In

this synthetic strategy, it is used in the protection of the anomeric hydroxyl, because it is a very

stable group, and it is only removed in certain conditions. The synthesis of 10 consists in the

deallylation of 9, using a described procedure [2]

with palladium (II) chloride as catalyst, and

methanol as reagent and solvent.

Palladium (II) chloride is the electrophile and reacts with the olefin from the allyl very

easily, forming a complex. Then, methanol acts like a nucleophile, and attacks the olefin,

provoking the departure of the leaving group, which in this case is the sugar itself. The proton

from methanol is released and protonates the anomeric hydroxyl. Allyl methyl ether is formed

after decomplexation and palladium (II) chloride is regenerated (Scheme 2.13).

Scheme 2.13 - Mechanism for the synthesis of 2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-

mannopyranose 10.


pretended compound with a yield of 75 %. In this case, the use of allyl ether protecting only one

hydroxyl allows its selective removal without affecting the other protecting groups, which will

be important in the next step.

21

2.1.1.8 (2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-mannopyranosyl)-trichlo-

roacetimidate 11 synthesis

Scheme 2.14 – Synthesis of (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-mannopyranosyl)-trichlo-

roacetimidate 11 with ix) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 10 minutes, 76 %.

The synthesis of 11, consists in the conversion of the anomeric hydroxyl group into a

trichloroacetimidate using DBU as catalyst and trichloroacetonitrile as reagent, added

sequentially and dichloromethane as solvent, all stirred at 0ºC. As it was said before, besides the

fact that the glycosyl donor must contain all the hydroxyls protected, the anomeric carbon needs

a leaving group. The use of the trichloroacetimidate group as LG in carbohydrate chemistry was

first developed by R. R. Schmidt,[5][14]

and since then it has been often used.

In this base catalysed reaction, DBU first deprotonates the anomeric hydroxy group,

which becomes more nucleophilic and attacks more easily the triple bond system present in the

electron deficient trichloroacetonitrile. Then, DBU is regenerated, because it is deprotonated, to

give the proton to the leaving group. The reason why the final product is only the α anomer, is

due to the anomeric effect (thermodynamically the α anomer is more stable). In this way, the

anomeric oxygen atom has been transformed into a good leaving group.


pretended compound with a yield of 76 % and α +38.8 (c 0.95, CH2Cl2). This synthesis was

successful. This glycosyl donor can be used in the synthesis of both disaccharides.

2.1.2 Monosaccharide glycosyl acceptors synthesis

For the formation of the disaccharides 1 and 2 two different glycosyl acceptors were

needed.

α-methyl-D-mannose was used as starting material for the preparation of the first

glycosyl acceptor. An efficient synthetic route for the synthesis of a 3-O methyl-mannose

glycosyl acceptor has been reported also by Liao et al[2]

. Once again, benzyl ether was used as

the protecting group at 2-OH. In this case, the acetyl group was chosen as protecting group at

2-OH not because it could influence the stereochemistry of the disaccharide but because of the

22

fact that the conditions used above for the acetylation gave very good yields. A synthetic

strategy based on the reported work [2]

was drawn with some additional changes. The proposed

synthetic route is shown below in Scheme 2.15.

Scheme 2.15 – Synthetic strategy followed for the synthesis of the glycosyl acceptor 16. Reagents and

conditions: i) benzaldehyde dimethyl acetal, camphorsulfonic acid, THF, Δ, overnight, 50 %;

ii) dibutyltin oxide, methanol, Δ, overnight and iii) iodomethane, DMF, 65 ºC, overnight, 2 steps: 50%;

iv) acetic anhydride, DMAP, 0ºC → rt, pyridine 2 hours; 97%; v), sodium cyanoborohydride, hydrogen

chloride in diethyl ether 1 M, THF, 0ºC, 100 %.

This strategy is very similar to the previous one, mostly because of the necessity of a

regioselective methylation step and the regioselective benzylidene opening step.

The second acceptor has already been synthesized, which is compound 8. The strategy

for the synthesis of glycosyl donor 11 (Scheme 2.1) was also drawn so that this intermediate

could be obtained.

2.1.2.1 Methyl 4,6-O-benzylidene-α-D-mannopyranoside 12 synthesis

Scheme 2.16 – Synthesis of methyl 4,6-O-benzylidene-α-D-mannopyranoside 12 with i) benzaldehyde

dimethyl acetal, camphorsulfonic acid, THF, Δ, overnight, 50 %.

The synthesis of 12 consists in a regioselective formation of a 4,6-O benzylidene acetal,

using the same conditions for the synthesis of 4, but with the reaction time increased to

overnight, because the compound is more polar and takes longer to dissolve in THF.


pretended compound with a yield of 50 %. This yield was not the expected for this reaction,

which may be due to a problem of solubility of the starting material in THF. Since the reaction

did not occur using DMF as solvent (a more polar solvent) and the use of benzylidene acetal as

a protecting group in this synthesis is important, this result was accepted.

23

2.1.2.2 Methyl 4,6-O-benzylidene-3-O-methyl-α-D-mannopyranoside 14 synthesis

Scheme 2.17 – Synthesis of methyl 4,6-O-benzylidene-3-O-methyl-α-D-mannopyranoside 14 with ii)

dibutyltin oxide, methanol, Δ, overnight and iii) iodomethane, DMF, 65 ºC, overnight; 2 steps: 50%.

The synthesis of 14 consists in a regioselective 3-O methylation. The conditions used

for the synthesis of 6 were applied to 12.


pretended compound with a yield of 33%. This yield was very low, comparing to the expected

for this reaction, which can be related to the fact that the compound is more polar, and needs

more reaction time and temperature to dissolve. In order to optimize it, the reaction time for ii)

was increased to overnight, and for iii) the reaction temperature was increased to 65 ºC.

Interpretation of the 1H-NMR spectrum revealed that the obtained product was the pretended

compound with a yield of 50 % (70% each step). In spite of the yield being better, it still was

not the expected one. Even so, this methylation step is very important for the synthetic route and

this result was acceptable.

The reason why, once again this synthetic strategy did not start with the methylation of

α-methyl-D-mannose, as it was reported by Liao et al[2]

, is going to be explained further in this

work.

2.1.2.3 Methyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-α-D-mannopyranoside 15

synthesis

Scheme 2.18 – Synthesis of methyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-α-D-mannopyranoside 15

with iv) acetic anhydride, DMAP, 0ºC → rt, pyridine 2 hours; 97%.

The synthesis of 15 consists in an acetylation of the 2-OH, using the conditions for the

synthesis of 7. Interpretation of the 1H-NMR spectrum revealed that the obtained product was

the pretended compound with a yield of 97%. This group will not influence the stereochemistry

of the disaccharide, but the hydroxyl needed to be protected, so the acetyl group was a good

choice, due to the high yield obtained.

24

2.1.2.4 Methyl 2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 16

synthesis

Scheme 2.19 – Synthesis of methyl 2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 16 with

v), sodium cyanoborohydride, hydrogen chloride in diethyl ether 1 M, THF, 0ºC, 100 %.

The synthesis of 16 consists in a regioselective reduction opening of the benzylidene

acetal using the conditions for the synthesis of 8. Interpretation of the 1H-NMR spectrum

revealed that the obtained product was the pretended compound with a yield of 100 %. The

synthesis of this glycosyl acceptor was successful.

2.1.3 Glycosylation reaction and hydroxyl group deprotection

After the synthesis of the glycosyl donor and acceptors, the respective monosaccharides

are ready for the glycosylation reaction. The glysosyl donor 11 has a leaving group in its

anomeric carbon, and the other hydroxyls are all protected. The glycosyl acceptors 16 and 8

have all the hydroxyls protected, except for the 4-OH. After the formation of the disaccharide,

the protecting groups have to be removed from the molecule. The deprotection steps have to be

very efficient, and must not hydrolyze the molecule.

A synthetic route for the glycosylation reaction and further protecting group removal

was proposed for the first disaccharide (Scheme 2.20).

Scheme 2.20 – Synthetic route followed for the synthesis of the disaccharide 1. Reagents and conditions:

i) TMSOTf, dichloromethane, -20 ºC, 30 minutes, 69 %; ii) sodium methoxide, methanol, rt, 2 hours: 30

minutes, 98%; iii) H2/Pd/C 10%, ethyl acetate /ethanol 1:1, 50 psi, overnight, 100 %.

25

2.1.3.1 Methyl (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-

(1→4)-2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 17 synthesis

Scheme 2.21 – Synthesis of methyl (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-

(1→4)-2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 17 with i) TMSOTf, dichloromethane,

-20 ºC, 30 minutes, 69 %.

The synthesis of 17 consists in a glycosylation reaction between 11 and 16, using

TMSOTf as catalyst and dichloromethane as solvent, all stirred at -20ºC. As it was said before,

most chemical glycosylation reactions need a catalyst, a promoter, to assist the departure of the

leaving group. This catalyst can be either a Lewis or a Brønsted acid.[5]

The use of O-glycosyl trichloroacetimidate donors has many advantages, such as the

fact that they are easily prepared, sufficiently stable, the use of heavy metal salts as promoters

can be avoided and they can be activated with catalytic amounts of Lewis acids, such as

TMSOTf or BF3.OEt2.[14][15] The activation of this LG is initiated by coordination of TMSOTf

to the nitrogen of the group. This LG now has conditions to depart from the molecule, using the

oxygen adjacent to the anomeric carbon as driving force. The formation of the oxonium ion is

followed by the attack of the acetate carbonyl oxygen to the anomeric carbon (neighbouring

group participation) to form an intermediate, the acyloxonium ion. The glycosyl acceptor has

now conditions to attack the intermediate to form a glycosidic bond. After the formation of the

disaccharide, the proton liberated on the glycosidic bond formation reacts with the forming

leaving group. The Lewis acid is released, becomes available for the next catalytic cycle, and

also trichloroacetamide is formed (Scheme 2.22).[5][15]

26

Scheme 2.22 - Mechanism for the glycosylation reaction and synthesis of 17.

Interpretation of the 1H-NMR spectrum revealed only one doublet signal at δ 5.23 ppm,

corresponding to an anomeric proton, which is the α anomeric proton from the glycosidic bond,

due to the effect of the participating group. The absence of the signal corresponding to the β

anomeric proton, indicates that the obtained product was the pretended compound, and not a

mixture of anomers. A disaccharide with a yield of 69 % was obtained. After confirming that

the obtained disaccharide was the pretended product the specific rotation of the compound was

measured and α +50.4 (c 1.04, CH2Cl2) was obtained. Despite the solvent used in this

reaction being dichloromethane, the main reason for this reaction to be stereoselective was due

to the use of the acetate group at 2-OH, which strongly favors the formation of the α-glycosidic

bond. After the formation of the disaccharide the protecting groups must be removed from the

molecule.

27

2.1.3.2 Methyl (6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-6-O-benzyl-3-

O-methyl-α-D-mannopyranoside 18 synthesis

Scheme 2.23 - Synthesis of methyl (6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-6-O-benzyl-3-

O-methyl-α-D-mannopyranoside 18 with ii) sodium methoxide, methanol, rt, 2 hours: 30 minutes, 98%.

After the formation of the glycosidic bond, it is important to find methods to remove the

protecting groups on both monosaccharide precursors, without degrading the molecule. The

synthesis of 18 consists in the deacetylation of 17, using sodium methoxide as catalyst, and

methanol as solvent.

The reaction first starts with a nucleophilic addition to the carbonyl from the acetate

group by the methoxide ion, followed by the departure of the sugar, which becomes deprotected

in that alcohol. With the formation of methyl acetate, there is not a possibility to regenerate the

catalyst from this compound. Besides the fact that methanol is the solvent of the reaction, it

also has an important role in the regeneration of the catalyst. Methanol is deprotonated and the

methoxide ion is regenerated.


pretended compound with a yield of 98% and a α +55.4 (c 0.95, CH2Cl2). This yield reveals

that removing first the acetyl groups from the molecule was a good choice.

2.1.3.3 Methyl (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-D-

mannopyranoside 1 synthesis

Scheme 2.24 – Synthesis of methyl (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-D-

mannopyranoside 1 with iii) H2/Pd/C 10%, ethyl acetate /ethanol 1:1, 50 psi, overnight, 100 %.

The synthesis of 1 consists in the hydrogenation of 18, in order to completely

debenzylate the molecule, using Pd/C 10% as catalyst, and a ethyl acetate/ethanol 1:1 as a

28

mixture of solvents, shaken at 50 psi of hydrogen. One of the advantages of this hydrogenation

method is the fact that the obtained product comes very pure, and a purification is not needed,

so high yields can be obtained.


pretended compound with a yield of 100 % and α +67.5 (c 0.99, H2O). This very good yield

also indicates that first removing the acetyls and then the benzyl ether groups was a good

choice. The synthesis of 1 was successful and efficient.

A route for the glycosylation reaction and further protecting group removal was

proposed for the second disaccharide (Figure 2.25).

Scheme 2.25 – Synthetic route followed for the synthesis of the disaccharide 2. Reagents and conditions:

i) TMSOTf, dichloromethane, -20 ºC, 30 minutes, 77 %; ii) palladium (II) chloride, methanol, rt, 2 hours;

80%; iii) sodium methoxide, methanol, rt, 6 hours: 30 minutes, 78%; iv) H2/Pd/C 10%, ethyl

acetate /ethanol 5:1, 50 psi, 7 hours, 98 %.

2.1.3.4 Allyl (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-

2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 19 synthesis

Scheme 2.26 – Synthesis of allyl (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-

2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 19 with i) TMSOTf, dichloromethane, -20

ºC, 30 minutes, 77 %.

The synthesis of 19 consists in a glycosylation reaction between 11 and 8, using the

same conditions as in the synthesis of 17.

29

Interpretation of the 1H-NMR spectrum revealed the presence of two doublet signals at

δ 4.86 and 4.83 ppm corresponding to the anomeric protons from the allyl ether end, and a

multiplet signal (δ 5.25-5.21 ppm) which contains the peak for the α anomeric proton from the

newly formed glycosidic bond. Once again, the absence of the signal corresponding to the β

anomeric proton from the glycosidic bond, indicates that the obtained product was the pretended

compound. A disaccharide with a yield of 77 % (αα:αβ 9:1) was obtained. After confirming that

the obtained disaccharide was the pretended product the specific rotation of the compound was

measured and α +35.1 (c 1.05, CH2Cl2) was obtained. Once again the influence of the

acetate at 2-OH was very important for the stereochemistry of the final compound.

2.1.3.5 (2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-2-O-

acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose 20 synthesis

Scheme 2.27 – Synthesis of (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-2-O-

acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose 20 with ii) palladium (II) chloride, methanol, rt, 2

hours; 80%.

Since the pretended disaccharide 19 has a free hydroxyl group in its reducing end, 8

could be used as the glycosyl acceptor in the glycosylation reaction. The allyl ether needed to be

removed after the glycosylation reaction. The synthesis of 20 consists in the deallylation of 19,

using the same conditions as in the synthesis of 10.


pretended compound with a yield of 80 % (αα:αβ 10:1) and α +39.8 (c 0.98, CH2Cl2).

2.1.3.6 (6-O-Benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-6-O-benzyl-3-O-

methyl-(α/β)-D-mannopyranose 21 synthesis

Scheme 2.28 – Synthesis of (6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-6-O-benzyl-3-O-

methyl-(α/β)-D-mannopyranose 21 with iii) sodium methoxide, methanol, rt, 6 hours: 30 minutes, 78%.

30

The synthesis of 21 consists in the deacetylation of 20 using the same conditions as in

the synthesis of 18, but with a longer reaction time. The work-up had to be different also, due to

the fact that the product was more polar than 18. Dowex-H+

resin was added until neutral pH, so

that the methoxide ion could be protonated, to form methanol, which is then evaporated,

affording the pure product.


pretended compound with a yield of 78 % (αα:αβ 10:1) and a α +51.8 (c 0.95, CH2Cl2).

2.1.3.7 (3-O-Methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-(α/β)-D-mannopyra-

nose 2 synthesis

Scheme 2.29 - Synthesis of (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-(α/β)-D-

mannopyranose 2 with iv) H2/Pd/C 10%, ethyl acetate /ethanol 5:1, 50 psi, 7 hours, 98 %.

The synthesis of 2 consists in the hydrogenation of 21, in order to debenzylate the

molecule, using Pd/C 10% as catalyst, and ethyl acetate/ethanol 5:1 as mixture of solvents,

shaken at 50 psi of hydrogen.


pretended compound with a yield of 98% (αα:αβ 2:1) and α +57.4 (c 0.96, MeOH). The

synthesis of 2 was successful and efficient.

Since 2 has a free anomeric hydroxyl group, in solution this stereocenter can be

interconverted in both anomeric forms due to mutarotation. 2 is found as a mixture of anomers.

2.2 Tetrasaccharide synthesis

The third and last of the proposed objectives was the synthesis of a tetrasaccharide,

methyl (3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyranosyl-(1→4)-3-

O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyranoside 22 (Figure 2.3).

31

Figure 2.3 – The structure of methyl 3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-

mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyranoside 22.

For this synthesis, two disaccharides are needed, a disaccharide glycosyl donor and an

acceptor (Figure 2.4).

Figure 2.4 – The structure of the glycosyl donor (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-α-D-

mannopyranosyl-(1→4)-2-O-acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-mannopyranosyl)-

trichloroacetimidate 23 and the glycosyl acceptor methyl (2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-

mannopyranosyl)-(1→4)-2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 24.

The synthetic strategies for the preparation of the disaccharide glycosyl donor and

acceptor were planned.

2.2.1 Disaccharide glycosyl acceptor synthesis

In order to obtain the disaccharide glycosyl acceptor, a glycosidic reaction between two

mannose precursors was needed, as shown in Scheme 2.30.

Scheme 2.30 – Synthetic route followed for the synthesis of the disaccharide glycosyl acceptor 24.

Reagents and conditions: a) TMSOTf, dichloromethane, -20 ºC, 30 minutes.

One of the advantages of having previously synthesized the disaccharide precursors is

the use of some of its intermediates in the synthesis of this molecule. Intermediate 16 was used

as glycosyl acceptor for this reaction, due to the fact that the final disaccharide is methylated on

the α anomeric position and at 3-OH, and it has a free hydroxyl at 4-OH. The glycosyl donor

32

had to be synthesized because the final disaccharide precursor has a free hydroxyl, and there is

not a selective method to remove one acetyl group from disaccharide 17, at the pretended

position.

A synthetic strategy for the synthesis of the glycosyl donor, using a different protecting

group at 4-OH was proposed (Scheme 2.31).

Scheme 2.31 – Synthetic route proposed for the synthesis of the glycosyl donor 32. Reagents and

conditions: i) DIPEA, TBDMSOTf, dichloromethane, 0 ºC, 20 minutes, 89%.

2.2.1.1 Allyl 2-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-(α/β)-D-

mannopyranoside 25 synthesis

Scheme 2.32 - Synthesis of allyl 2-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-(α/β)-D-

mannopyranoside 25 with i) DIPEA, TBDMSOTf, dichloromethane, 0 ºC, 20 minutes, 89%.

The synthesis of 25 consists in the silylation of 8, using DIPEA, TBDMSOTf and

dichloromethane as solvent, stirred at -20ºC.

In this reaction, DIPEA first deprotonates the 4-OH, which becomes more nucleophilic,

attacks more easily the silicon atom of TBDMSOTf and the triflate group departs from the

molecule. DIPEA also neutralizes the triflic acid formed, which could remove the TBDMS

group from 25.


pretended compound with a yield of 89%. The use of this silyl ether as protecting group at

4-OH has great advantages, such as the fact that it is easily inserted on the molecule and can be

selectively removed in the presence of the other protecting groups. In this case, this group is

removed from the molecule, after the glycosylation reaction, allowing the formation of

compound 24.

33

2.2.1.2 Attempted synthesis of 2-O-Acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-

3-O-methyl-(α/β)-D-mannopyranose 26

Scheme 2.33 – Attempted synthesis of 2-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-

(α/β)-D-mannopyranose 26 with ii) bis(dibenzylideneacetone)palladium (0), 1,4-Bis (diphenyl-

phosphino)butane, THF, rt, 15 minutes and iii) 1,3-dimethylbarbituric acid, THF, 60 ºC, overnight.

The conditions used for the synthesis of 10 could not be used in the synthesis of 26, due

to the acidic conditions of the reaction medium when methanol is deprotonated, which can

remove the TBDMS group from the molecule. An alternative method for the deallylation of 25

was proposed.

This method consists in first activating bis(dibenzylideneacetone)palladium (0) to

palladium(II) using 1,4-Bis(diphenylphosphino)butane in dry THF, all stirred at room

temperature and then add it to a solution of 25 and 1,3-dimethylbarbituric acid in THF at the

same temperature. After the formation of the complex between palladium(II) and the allyl

group, 1,3-dimethylbarbituric acid, acts as a nucleophile just like methanol in the synthesis of

10, attacks the olefin, promoting the departure of the sugar. In this case, protons are not released

in the reaction medium, so there is less hypothesis for the silyl ether to be cleaved.

Using these conditions the starting material was not consumed after 30 minutes. The

reaction temperature was increased to 60ºC and stirred for another 30 minutes. The starting

material was not consumed, so the reaction time was increased to overnight. Even after

overnight the starting material was not consumed. Other methods for the removal of the allyl

group were tried.




(α/β)-D-mannopyranose 26 with iv) sodium borohydride, iodine, THF, 0 ºC, 3 hours and 20 minutes.

34

A method used in carbohydrates for the deallylation of 25 was the use of sodium

borohydride and iodine, added sequentially at 0ºC in THF.[16]

In this reaction, oxidation of this

reagent with iodine in THF gives BH3-THF, which can reduce the olefin from the allyl ether:

Scheme 2.35 - Mechanism for the synthesis of 2-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-

methyl-(α/β)-D-mannopyranose 26 using the described reaction conditions.[16]

However, using the described conditions, after stirring the mixture for 20 minutes the

starting material was not consumed. The mixture was stirred for more 3 hours and changes were

not observed.




(α/β)-D-mannopyranose 26 with v) t-BuOK, DMF, 60 ºC, 1 hour.

Another way to remove this protecting group is by isomerization, allowing the

formation of a prop-l-enyl group, which can be removed easily, using non-acidic conditions.[17]

A described method[18]

was applied to 25, using t-BuOK in DMF at 60ºC. This reagent is a very

strong base and is able to deprotonate the carbon adjacent to the double bond, allowing the

isomerization. After the formation of the prop-l-enyl group, a non-acidic method could be

performed for its removal, using iodine in THF/H2O.

The reaction was stirred for 1 hour. Interpretation of the 1H-NMR spectrum of the

reaction mixture revealed that the obtained product was not the expected compound. Since the

obtained compound was not the expected one, the step following the isomerization was not

used. Other methods for the deallylation of this compound were attempted.

35




(α/β)-D-mannopyranose 26 with vi) acetic acid/H2O (90 % v/v), sodium acetate, palladium (II) chloride,

ethyl acetate, rt, overnight.

Another interesting method which is employed for the deallylation of carbohydrates,

uses palladium (II) chloride, and a buffer solution (acetic acid/sodium acetate).[18]

The buffer

maintains the pH of the reaction medium constant, avoiding the removal of the silyl group. The

other differences between this method and the one using palladium (II) chloride in methanol is

the heterogeneous medium (H2O/ethyl acetate), and a non-catalytic amount of palladium (II)

chloride.

Before the formation of the complex, the acetic acid will act like an acidic catalyst and

will first protonate the oxygen from the allyl ether. After the formation of the complex, instead

of methanol, H2O acts like a nucleophile and attacks the olefin, promoting the departure of the

sugar and the formation of the hydroxyl group. The liberated proton will not induce the removal

of the silyl group due to the presence of the acetate ion.

This described method was applied to 25, with acetic acid/H2O (90% v/v), sodium

acetate and palladium (II) chloride being added sequentially at room temperature, and the

reaction mixture was stirred overnight. The starting material was totally consumed. After the

purification of the reaction mixture, interpretation of the 1H-NMR spectrum revealed that the

TBDMS group was still on the molecule and the signals of the allyl group disappeared.

However, there was a difficulty in interpreting the 1H-NMR spectrum, mainly because of two

extra doublets, which appeared at δ 4.24 ppm and 4.13 ppm, and what it seems to be a mixture

of two pairs of doublets (between δ 4.65 and 4.50 ppm) instead of the former ABdd at δ 4.60

ppm (in compound 25), corresponding to the protons from the CH2 of the benzyl groups (Figure

2.5).

36

Figure 2.5 –1H-NMR spectrum, corresponding to the mixture of two pairs of doublets (between δ 4.65

and 4.50 ppm) and two doublets (at δ 4.24 ppm and 4.13 ppm).

The 1H-NMR spectrum indicated a mixture of different compounds. In order to better

identify and characterize the products, the sample was acetylated following the same procedure

used in the preparation of compounds 7, 9 and 15. Two different products were obtained. One of

the products was 27, which is the pretended compound 26 acetylated, but only the α anomer

(Figure 2.6). In this spectrum one ABdd at δ 4.59 ppm was present, corresponding to the

protons from the CH2 of the benzyl groups (Figure 2.7). The signal corresponding to the

anomeric proton, due to the presence of the acetate group, is located at δ 6.03 ppm (Figure 2.7).

Figure 2.6 – The structure of 1,2-di-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-α-D-

mannopyranose 27.

In the other obtained product 28, interpretation of the 1H-NMR spectrum revealed one

ABdd at δ 4.58 ppm and the presence of the two doublets at δ 4.24 and 4.13 ppm. The signal of

the anomeric proton is located at δ 4.86 ppm (Figure 2.7), which reveals that the compound has

not been acetylated at 1-OH. Interpretation of the 13

C-APT spectrum revealed a signal at δ

204.79 ppm (Figure 2.8), which indicates the presence of a ketone or an aldehyde in the

molecule.

37

Figure 2.7 – 1H-NMR spectra overlay from the two obtained compounds (between δ 6.2 and 4.0 ppm).

The green spectrum is from compound 27 and the red one is from compound 28.

Figure 2.8 – 13

C-APT spectra overlay from the two obtained compounds (between δ 220.0 and -20.0

ppm). The green spectrum is from compound 27 and the red one is from compound 28.

28

27

28

RCOR or RCOH

-OCH2Ph, ABdd, 2H

two doublets

-OCH2Ph, ABdd, 2H

H-1 (anomeric proton), d, 1H

H-1 (anomeric proton), d, 1H

27

38

A reported work by Lüning at al[19]

indicates that the deallylation using these conditions

affords a byproduct, which is the formation of the Wacker oxidation product on the allyl group.

With this oxidation of the olefin, two products can be formed:

Figure 2.9 – Two possible products, which result from the Wacker oxidation of the allyl group.

With the interpretation of the NMR data and its comparison with the reported work by

Lüning at al, it can be concluded that compound 28 results from the Wacker oxidation. In this

case, the ketone was formed (a), because the two doublets at δ 4.24 and 4.13 ppm correspond to

the protons from the CH2 adjacent to the carbonyl. Besides that, the singlet signal from the other

three protons adjacent to the ketone appear at δ 2.15 or at 2.10 ppm. The formation of this

Wacker product brought some disadvantages, because it was impossible to separate the two

compounds, without having to acetylate them. Besides that, the yield for the formation of 26

was low (48%), and for the Wacker product was 29%. The mechanism for this reaction is

shown in scheme 2.38.

Scheme 2.38 – Mechanism for the Wacker oxidation.

The catalyst needed a certain quantity of oxidant (for example CuCl2) to be regenerated.

However since the quantity of palladium (II) chloride added is stoichiometric this was not

necessary. Once again, only the α anomer was formed.

In order to avoid the formation of the Wacker product and to optimize this method,

different reaction times and reagent quantities (PdCl2) were studied.

a b

39

Table 2.1 – Summary of the different experimental conditions used for the optimization of the synthesis

of 26.

Reaction

time

Quantity of

PdCl2

Starting

material 26

28 (Wacker

product)

Normal

conditions Overnight 1.5 equivalents No Yes Yes

1 6 hours 1.5 equivalents Yes Yes Yes



Shorter reaction times did not afford good results as well, neither less quantity of

palladium (II) chloride. Since it was impossible to synthesize 26 without the parallel formation

of 28 another method for the allylation was attempted.



Scheme 2.39 - Attempted synthesis of 2-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-

(α/β)-D-mannopyranose 26 with vii) (dimethyl sulfide)trihydroboron, THF, 0 ºC, 20 minutes.

For the deallylation of 25 (dimethyl sulfide)trihydroboron, added at 0ºC in THF was

tried.[16]

This method is similar to the one used previously with sodium borohydride and iodine,

but in this case the reagent does not need to be activated by the oxidation of iodine, since it is

already in the BH3 form. After 20 minutes, the starting material was totally consumed.

However, the formation of several products was observed. Interpretation of the 1H-NMR

spectrum from the different products revealed none of them was the expected compound.

40

Since an efficient method to remove the allyl group was not found, other alternatives

were considered. Another synthetic strategy was proposed:

Scheme 2.40 – Alternative synthetic route proposed for the synthesis of the glycosyl donor 32. Reagents

and conditions: i) acetic acid/H2O (90 % v/v), sodium acetate, palladium (II) chloride, ethyl acetate, rt,

overnight 73%; ii) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 10 minutes, 36 %; iii) DIPEA,

TBDMSOTf, dichloromethane, 0 ºC, 20 minutes, 74 %.

2.2.1.7 2-O-Acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose 29 synthesis

Scheme 2.41 – Synthesis of 2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose 29 with

i) acetic acid/H2O (90 % v/v), sodium acetate, palladium (II) chloride, ethyl acetate, rt, overnight 73%.

In this reaction, in order to avoid the obstacles verified on the previous route, 8 was

deallylated before being silylated at the 4-OH.

Using the conditions for the synthesis of 10, interpretation of the 1H-NMR spectrum

indicated that the pretended compound was obtained, but with a yield of 22 %. This yield was

not the expected and it was very low, so the other conditions using palladium (II) chloride and

the buffer solution were experimented.[18]

Compound 29 was obtained with a yield of 73%.

2.2.1.8 (2-O-Acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-mannopyranosyl)-trichloro-

acetimidate 30 synthesis

Scheme 2.42 – Synthesis of (2-O-acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-mannopyranosyl)-trichloro-

acetimidate 30 with ii) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 10 minutes, 36 %.

The synthesis of 30 should consist in a regioselective trichloroacetimidation at the

anomeric hydroxyl group. To accomplish that, a reported procedure[20]

was applied for the

synthesis of 30, but instead of using Cs2CO3 as base, DBU was used. The difference between

41

this procedure and the one used in the synthesis of 11 is the different quantity of base and

trichloroacetonitrile. In this case, less quantity of both reagents was needed, in order to avoid

the trichloroacetimidation on both hydroxyls. However 10 minutes later the formation of two

products was observed.

Figure 2.10 – The structure of (2-O-acetyl-6-O-benzyl-3-O-methyl-1,4-O-α-D-mannopyranosyl)-di-

trichloroacetimidate 31.

Interpretation of the 1H-NMR spectrum revealed that the obtained products were the

pretended compound 30, with a yield of 36% and 31 (Figure 2.10), with a yield of 59%. The

yield for 30 unfortunately was not the expected, even with the changed conditions. However, 30

will be used further in this work in the synthesis of 32.

2.2.1.9 (2-O-Acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-1-O-α-D-

mannopyranosyl)-trichloroacetimidate 32 synthesis

Scheme 2.43 – Synthesis of (2-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-1-O-α-D-

mannopyranosyl)-trichloroacetimidate 32 with iii) DIPEA, TBDMSOTf, dichloromethane, 0 ºC, 20

minutes, 74 %.

Despite the yield obtained in the synthesis of 30 being very low, due to the formation of

31, it could be possible that this reaction could be optimized by changing the parameters of the

procedure, avoiding the formation of the secondary product.

30 was silylated at 4-OH in order to see if the previous reaction is the only one that

needs to be optimized. Interpretation of the 1H-NMR spectrum revealed that the obtained

product was the pretended compound 32, with a yield of 74 %. With this result, it can be

concluded that this route could be successful if the previous trichloroacetimidation was a more

efficient reaction.

42

2.2.1.10 Methyl (2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-

2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 24 synthesis

Scheme 2.44 - Synthesis of methyl (2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-

2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 24 with iv) TMSOTf, dichloromethane,

-20 ºC, 30 minutes, 18 %.

This alternative step consists in using 31 as the glycosyl donor in the glycosylation

reaction to synthesize 24. The same conditions as in the synthesis of 17 and 19 were used. If

this reaction afforded a good yield, the regioselective step to synthesize 30 would not be needed.

In this synthesis, after the glycosylation reaction, the trichloroacetimidate group protecting the

4-OH departs from the molecule, since it is a very unstable group and hydrolyses very easily.

However, interpretation of the 1H-NMR spectrum revealed that the obtained product

was the pretended compound 24, but with a low yield of 18%, which revealed that 31 was not a

good glycosyl donor. Other possibilities were studied.

Since this synthetic strategy (Scheme 2.40) did not go as planned, another alternative

was proposed:

Scheme 2.45 – Synthetic route proposed for the synthesis of the glycosyl donor 34. Reagents and

conditions: i) acetic acid/H2O (90 % v/v), sodium acetate, palladium (II) chloride, ethyl acetate, rt,

5 hours, 78%; ii) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 3 hours, 10%.

Instead of using compound 32, 34 could be used as glycosyl donor. After the

glycosylation reaction, the benzylidene group could be reduced to afford 24.

43

2.2.1.11 2-O-Acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranose 33

Scheme 2.46 – Synthesis of 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranose 33 with

i) acetic acid/H2O (90 % v/v), sodium acetate, palladium (II) chloride, ethyl acetate, rt, 5 hours, 78%.

The synthesis of 33 consists in the deallylation of 7, using palladium (II) chloride and

the buffer solution[18]

, with the reaction time decreased to 5 hours. The conditions used for the

synthesis of 10, palladium (II) chloride and methanol, were not applied in this synthesis due to

the presence of the benzylidene acetal, which is removed under acidic conditions.


pretended compound, with a yield of 78 %.

2.2.1.12 (2-O-Acetyl-4,6-O-benzylidene-3-O-methyl-1-O-α-D-mannopyranosyl)-tri-

chloroacetimidate 34

Scheme 2.47 – Synthesis of (2-O-acetyl-4,6-O-benzylidene-3-O-methyl-1-O-α-D-mannopyranosyl)-tri-

chloroacetimidate 34 with ii) DBU and trichloroacetonitrile, dichloromethane, 0ºC, 3 hours, 10 %.

The synthesis of 34 consists in the trichloroacetimidation of 33, using the same

conditions as in the synthesis of 11, but with a longer reaction time.

However, interpretation of the 1H-NMR spectrum revealed that the obtained product

was the pretended compound, with a very low yield of 10 %. With this result this route was not

a good alternative.

One of the main obstacles in the synthesis of the glycosyl donor 32 was the use of allyl

ether as protecting group at 1-OH. Even though the allyl ether is one of the most used protecting

groups in carbohydrate chemistry, the fact that it is removed only under certain conditions

brings some disadvantages when using other protecting groups, such as for example silyl ethers.

44

What if in this synthesis, another protecting group could be used at the anomeric

position? The acetyl group for example could be a very good protecting group, since it is easily

inserted and removed under some conditions which cannot remove other protecting groups or

form secondary products. The only obstacle in the use of this protecting group, is that it has to

be used also at 2-OH, due to the neighbouring group participation. So, a procedure for the

regioselective removal of the acetyl group at the anomeric position has to be applied. A new

strategy was proposed for the synthesis of glycosyl donor 32 (Scheme 2.48).

Scheme 2.48 – Alternative synthetic route followed for the synthesis of the glycosyl donor 32.

A reported work[21]

used α-methyl-D-mannose as starting material for the synthesis of

3-O methyl mannose, such as Liao and coworkers.[2]

However this work[21]

described a method

to remove the anomeric methoxy group, by using a mixture of reagents (acetic anhydride, acetic

acid and sulfuric acid) to afford an aggressive acetylation step, which can be useful in the

proposed strategy. The reason why the benzylidene group has to be inserted in the molecule

afterwards is due to the fact that it would be removed from the molecule with the aggressive

acetylation step. Then, after 1-OH and 2-OH acetylation, the benzylidene ring can be

regioselectively opened, in order to allow the 4-OH TBDMS protection. After the silyl ether

protection, since there are two acetyl groups, and only one needs to be removed, some

procedures can be used to accomplish that, such as the use of hydrazine acetate. [22]

2.2.1.13 Attempted synthesis of Methyl 3-O-methyl-α-D-mannopyranoside 35

Scheme 2.49 – Attempted synthesis of methyl 3-O-methyl-α-D-mannopyranoside 35 with i) dibutyltin

oxide, toluene, Δ, 3 hours; ii) iodomethane, TBAI, toluene, 70 ºC, 72 hours.

45

The reported 2 step method[21]

for the regioselective 3-O methylation was applied to α-

methyl-D-mannose using dibutyltin oxide on the first step and on the second step iodomethane

and TBAI as reagents, and toluene as solvent on both steps. TBAI can stabilize the iodine atom,

facilitating its departure from the iodomethane molecule, in order to increase the reaction rate.

However, interpretation of the 1H-NMR spectrum, revealed that the reaction did not

occur, probably because of a solubility problem. This starting material is a much more polar

compound, so its solubility in tolune is lower and the formation of the 2,3-O-di-butylstannylene

intermediate is not favored. Even if the stannylene intermediate is formed, this compound

hardly dissolved in the solvent and even with 72 hours of reaction time, the reaction with

iodomethane and TBAI is not favored.

2.2.1.14 Attempted synthesis of Methyl 3-O-methyl-α-D-mannopyranoside 35

Scheme 2.50 – Synthesis of methyl 3-O-methyl-α-D-mannopyranoside 35 with iii) dibutyltin oxide,

methanol, Δ, overnight; iv) iodomethane, DMF, 65 ºC, overnight.

The method applied on the synthesis of 14 was used in this step.

Once again, interpretation of the 1H-NMR spectrum, revealed that the reaction did not

occur, which also may be due to a solubility problem. The reason why during this work the

synthesis of glycosyl donor 11 and acceptor 16 started first with the benzylidenation and then

with the methylation step is mainly due to this result.

A way to solve this problem was to decrease the polarity of α-methyl-D-mannose by

protecting one or several hydroxyls of the molecule. A new strategy for the synthesis of 32,

which included this important step, was drawn and proposed (Scheme 2.51)

46

Scheme 2.51 – Alternative route proposed for the synthesis of the glycosyl donor 32. Reagents and

conditions: i) TrCl, pyridine, rt, 24 hours; ii) TrCl, DMAP, pyridine rt, overnight; 2 steps: 100 %;

iii) dibutyltin oxide, methanol, Δ, overnight and iv) iodomethane, DMF, 65 ºC, overnight; 2 steps : 68 %;

v) acetic anhydride/acetic acid/sulfuric acid 105:45:1, v/v/v, rt, overnight, 80%.

Trityl was chosen as the protecting group, because it can be inserted and removed from

the molecule very easily. This group will assist the 3-O methylation reaction and then will be

removed in the aggressive acetylation step.

2.2.1.15 Methyl 6-O-trityl-α-D-mannopyranoside 36 synthesis

Scheme 2.52 – Synthesis of methyl 6-O-trityl-α-D-mannopyranoside 36 with i) TrCl, pyridine, rt, 24

hours; ii) TrCl, DMAP, pyridine rt, overnight; 2 steps: 100 %.

The synthesis of 36 consists in the tritylation of α-methyl-D-mannose, using a two step

reaction,

In the first step TrCl is kept at rt with pyridine, in order to allow the departure of the

chloride leaving group for the formation of the trityl carbocation. In the second step DMAP

forms an activated species with the carbocation. The 6-OH group attacks the carbon, DMAP

departs from the molecule, and 36 is formed. The catalyst is protonated but then is regenerated

by pyridine. The reason why this tritylation is 6-O regioselective is due to the fact that this

group is very bulky and selectively reacts with primary alcohols in carbohydrates.


pretended compound, with a yield of 100 %.

47

2.2.1.16 Methyl 3-O-methyl-6-O-trityl-α-D-mannopyranoside 37 synthesis

Scheme 2.53 – Synthesis of methyl 3-O-methyl-6-O-trityl-α-D-mannopyranoside 37 with iii) dibutyltin

oxide, methanol, Δ, overnight; iv) iodomethane, DMF, 65 ºC, overnight; 2 steps : 68 %.

The same conditions as in the synthesis of 14 were applied to 36 and interpretation of

the 1H-NMR spectrum revealed that the obtained product was the pretended compound, with a

yield of 68 %. The use of trityl group to decrease the polarity of the compound was a very good

choice, since the yield for the tritylation was very high (100 %) and the yield for the 3-O-

methylation step was good.

2.2.1.17 1,2,4,6-Tetra-O-acetyl-3-O-methyl-(α/β)-D-mannopyranose 38 synthesis

Scheme 2.54 – Synthesis of 1,2,4,6-tetra-O-acetyl-3-O-methyl-(α/β)-D-mannopyranose 38 with v) acetic

anhydride/acetic acid/sulfuric acid 105:45:1, v/v/v, rt, overnight, 80 %.

The synthesis of 38 consists in the acetylation of 37 using a described procedure[21]

, with acetic

anhydride and acetic acid as reagents and solvents, and sulfuric acid as catalyst, all stirred at rt.

Besides the acetylation of the free hydroxyl groups, the anomeric methoxy group can be

removed from the molecule when is protonated by the acidic catalyst, giving the formation of

the oxonium ion. After that, the acetate ion attacks the anomeric carbon on both sides of the

molecule, forming 38.


pretended compound, with a yield of 80 %. The rest of the synthetic strategy could not be

continued, but with these very good results, it is a very promising one.

48

2.2.2 Disaccharide glycosyl donor synthesis

One of the advantages of having synthesized first the disaccharide precursors was the

use of some of its intermediates in the synthesis of the tetrasaccharide. 20 could be used in the

synthesis of the disaccharide glycosyl donor, since it has a free anomeric hydroxyl group, ready

to be trichloroacetimidated:

Scheme 2.55 – Synthesis of the disaccharide glycosyl donor 23. Reagents and conditions: a) DBU and

trichloroacetonitrile, dichloromethane, 0ºC, 10 minutes.

However, since the glycosyl acceptor disaccharide 23 could not be synthesized, this

compound was not synthesized in this work.

49

CHAPTER 3

CONCLUSION

50

51

3. Conclusion

The three main objectives of this work were the efficient synthesis of three saccharides,

which are cellular precursors for the biosynthesis of MMPs.

The two first sugars are disaccharides and to synthesize them a glycosyl donor and

acceptor were needed. The same glycosyl donor 11 was used in the synthesis of both

disaccharides. D-mannose was used as starting material. The synthesis was efficient, with

individual yields equal or higher to 75 %, except for the benzylidenation step, which had a yield

of 59%. However, this step was very important in the synthesis, because this acetal can be

regioselectively opened and can facilitate the 3-O methylation, since it lowers the polarity of the

sugar. Different glycosyl acceptors were used in the synthesis of each disaccharide, since they

have structural differences - one has a reducing end and the other does not. The synthesis of the

first glycosyl acceptor 16 used α-methyl-D-mannose as starting material. This was successful

with individual yields higher than 95%, except for the benzylidenation and the methylation

steps, which had yields of 50%. Once again, the acetalation step is important in this synthesis,

so this reasonable yield was acceptable. The yield for the methylation step was also reasonable,

since it is a 2 step reaction (70% yield each step), and it is a very important step for the

synthesis. The second glycosyl acceptor 8 was one of the intermediates in the synthesis of the

glycosyl donor 11, so it was also successfully synthesized. The “building blocks” for the

formation of both disaccharides were ready for the glycosylation reaction.

The synthesis of the first disaccharide 1, using 11 and 16 as glycosyl donor and

acceptor, respectively, was successful. The glycosylation reaction afforded esclusively the α

anomer, which was the pretended product with a yield of 69%. The use of the acetyl group at 2-

OH of the glycosyl donor was a good strategy to induce the formation of the pretended anomer,

due to the participating group effect. The use of the trichloroacetimidate group as leaving group

was also a good choice, since the yield of the glycosylation was good. After the formation of the

glycosidic bond, the disaccharide was deprotected, to give the pretended compound 1. The

strategy used for the removal of the protecting groups was successful, with individual yields

higher or equal than 98%.

The synthesis of the second disaccharide 2, using 11 and 8 as glycosyl donor and

acceptor, respectively, was also successful. The glycosylation reaction afforded exclusively the

pretended α glycosidic bond, with a yield of 77 %. The use of the same reagents and the

participating group at 2-OH were important for the outcome of the glycosylation reaction in

terms of yield and stereoselectivity. Also, the removal of the protecting groups was successful,

with individual yields higher or equal than 78%. Since the configuration of the anomeric carbon

52

from the reducing end can be interconverted due to a process called mutarotation, it was not

relevant.

The third and last saccharide to be synthesised was a tetrasaccharide. Both disaccharide

donor 23 and acceptor 24 needed to be first synthesized. 24 could be obtained from the

glycosylation reaction between glycosyl donor 32 and the already synthesized acceptor 16. 32

needed to have a silyl group at 4-OH, so that after the glycosylation reaction this group could be

selectively removed, to form the disaccharide 24. However, removing the allyl group with the

molecule containing the 4-OTBDMS group was very difficult. The allyl group proved to be a

good protecting group in the synthesis of 11, but in the synthesis of 32 its constant use in

carbohydrate research was questioned. Despite being removed from the molecule under certain

conditions, sometimes those methods can form undesired secondary products. Some alternative

synthetic strategies were proposed.

One of the proposed synthetic routes (Scheme 2.40), which deallylates the sugar before

the formation of the silyl ether, gave good results, with individual yields higher or equal than

73%, except for the regioselective trichloroacetimidation step, which had a very low yield of

36%. Other alternative methods were attempted. In a future work, if some reactional conditions

are found to increase this yield, this could be a very promising route for the synthesis of 32.

Another promising strategy (Scheme 2.51), was proposed without the use of allyl ether

as protecting group. The acetyl group was used as alternative, since there are methods which

regioselectively remove this protecting group at the anomeric position. Unfortunately this

strategy could not be continued in this work due to lack of time, and had to be stopped in the

synthesis of 38 but with yields equal or higher than 68%. In a future work this strategy has great

potential for the efficient synthesis of 32.

Since the disaccharide 24 could not be formed, disaccharide 23 was not synthesized in

this work. However, in a future work compound 20 could be trichloroacetimidated, to form 23.

In general, this work had most of the objectives achieved. Even though the synthesis of

the tetrasaccharide was not complete, some helpful tools for its chemical formation were

developed. The reactions that did not go as the expected can guide a future work to not follow

those. This work also successfully highlighted the importance of chemical glycosylation, in

comparison with enzymatic glycosylation. It is very hard to purify a compound obtained from

an enzymatic reaction, and the enzymatic reactions are usually performed in small scale, as they

often need expensive co-factors. Moreover, the main objective of the synthesis of these

compounds is to discover the enzymes which catalyse the formation of these glycosidic bonds,

in order to characterize the synthesis of MMPs in vivo. So, since the enzymes which can

53

catalyse the formation of these saccharides have to be found, the only way to synthesize them is

by chemical glycosylation.

54

55

CHAPTER 4

EXPERIMENTAL PART

56

57

4. Experimental part

4.1 General conditions

All reactions were carried out under an inert atmosphere (argon), except when the solvents were

not dried. Air sensitive materials were handed in a Braun MB 150-Gl glove box. The

synthesized compounds were purified by silica flash column chromatography or silica

preparative TLC. Reactions were followed by Analytical TLC. The purity of synthesized

compounds was also verified with Analytical TLC and the characterization of the same

compounds was done by 1H-NMR,

13C-NMR,

13C-APT, 2D techniques (COSY and HMQC),

IR spectroscopy and specific rotation, when applicable.

Analytical TLC was performed on aluminium-backed Merck 60 F254 silica gel plates. The spots

corresponding to the products were identified by UV radiation (254 nm) and then immersed on

a 5% phosphomolybdic acid solution in ethanol.

Silica preparative TLC in Silica gel Merck 60 F254.

Silica flash column chromatography in Silica gel Merck 60.

1H-NMR spectra were recorded on a Bruker 400 spectrometer and obtained at 400 MHz in

CDCl3 or D2O. Chemical shifts are given in ppm, downfield from tetramethylsilane, for

solutions in CDCl3. Spectra in D2O are pre-saturated on the water signal (4.7 ppm).

13C-NMR spectra were recorded on a Bruker 400 spectrometer at 100.61 MHz in CDCl3 or

D2O.

IR spectra were measured on a Nicolet 6700 ATR-FTIR spectrometer with a Zn-Se crystal.

Specific rotations ([α]20

D) were measured on a Perkin-Elmer D241 automatic polarimeter at the

sodium D-line at 20 ºC, and reported as [α]D (concentration in g/100 mL of solvent).

4.2 Solvent and Reagent Purification

All the used solvents were previously distilled in the laboratory.

Acetic Anhydride: distilled under reduced pressure.

Allyl alcohol: distilled at atmospheric pressure.

DBU: distilled under reduced pressure.

58

Dry Dichloromethane: previously distilled DCM was stirred with phosphorous pentoxide

(drying agent) for 2 hours under reflux, being only distilled before its utilization.

DIPEA: distilled under reduced pressure, using calcium hydride as drying agent.

Dry DMF: to previously distilled DMF calcium hydride (drying agent) was added and the

mixture was left overnight, followed by decantation from the drying agent and distillation under

reduced pressure.

Dry Ethyl Ether: same procedure than THF and stored with sodium wire.

Dry Methanol: to 50-70 mL of previously distilled methanol 5g of magnesium turnings and

iodine (0.5 g) were added, and it was refluxed until all the magnesium had been consumed.

More methanol (1L) was added and the reflux was maintained for 2h.

Dry Pyridine: distilled twice at atmospheric pressure using potassium hydroxide as drying

agent.

Dry THF: to previously distilled THF, sodium wire and benzophenone were added, and the

mixture was refluxed under argon for several hours until the solvent turns deep blue in colour.

Then the mixture was kept at low reflux, being only distilled before its utilization.

TMSOTf: distilled at atmospheric pressure.

Dry Toluene: distilled at atmospheric pressure using sodium as drying agent, and stored with

sodium wire.

Trichloroacetonitrile: distilled at atmospheric pressure.

59

4.3 Compound list

Compound Nº Name Exp. Page

1

Methyl (3-O-methyl-α-D-

mannopyranosyl)-(1→4)-3-O-

methyl-α-D-mannopyranoside

15 75

2

(3-O-Methyl-α-D-


methyl-(α/β)-D-mannopyranose

19

79

3 Allyl (α/β)-D-mannopyranoside 1 65

4 Allyl 4,6-O-benzylidene-(α/β)-

D-mannopyranoside 2 65

6

Allyl 4,6-O-benzylidene-3-O-

methyl-(α/β)-D-

mannopyranoside

3 66

7

Allyl 2-O-acetyl-4,6-O-

benzylidene-3-O-methyl-(α/β)-

D-mannopyranoside

4 67

8

Allyl 2-O-acetyl-6-O-benzyl-3-

O-methyl-(α/β)-D-

mannopyranoside

5 68

9

Allyl 2,4-di-O-acetyl-6-O-

benzyl-3-O-methyl-(α/β)-D-

mannopyranoside

6 69

10

2,4-di-O-Acetyl-6-O-benzyl-3-

O-methyl-(α/β)-D-

mannopyranose

7

70

11

(2,4-di-O-Acetyl-6-O-benzyl-3-

O-methyl-1-O-α-D-

mannopyranosyl)-

trichloroacetimidate

8

70

Table 4.1: Summary table of the synthesized compounds.

60

12 Methyl 4,6-O-benzylidene-α-D-

mannopyranoside 9 71

14 Methyl 4,6-O-benzylidene-3-O-

methyl-α-D-mannopyranoside 10 72

15

Methyl 2-O-acetyl-4,6-O-

benzylidene-3-O-methyl-α-D-

mannopyranoside

11 72

16

Methyl 2-O-acetyl-6-O-benzyl-

3-O-methyl-α-D-

mannopyranoside

12 73

17

Methyl (2,4-di-O-acetyl-6-O-

benzyl-3-O-methyl-α-D-


acetyl-6-O-benzyl-3-O-methyl-

α-D-mannopyranoside

13 73

18

Methyl (6-O-benzyl-3-O-methyl-

α-D-mannopyranosyl)-(1→4)-6-

O-benzyl-3-O-methyl-α-D-

mannopyranoside

14 74

19

Allyl (2,4-di-O-acetyl-6-O-

benzyl-3-O-methyl-α-D-



(α/β)-D-mannopyranoside

16 76

20

(2,4-di-O-Acetyl-6-O-benzyl-3-

O-methyl-α-D-



(α/β)-D-mannopyranose

17 77

21

(6-O-Benzyl-3-O-methyl-α-D-


benzyl-3-O-methyl-(α/β)-D-

mannopyranose

18

78

61

22

Methyl 3-O-methyl-α-D-

mannopyranosyl-(1→4)-3-O-

methyl-α-D-mannopyranosyl-

(1→4)-3-O-methyl-α-D-

mannopyranosyl-(1→4)-3-O-

methyl-α-D-mannopyranoside

- -

23

(2,4-di-O-Acetyl-6-O-benzyl-

3-O-methyl-α-D-manno-

pyranosyl-(1→4)-2-O-acetyl-

6-O-benzyl-3-O-methyl-1-O-α-

D-mannopyranosyl)-


- -

24

Methyl (2-O-acetyl-6-O-benzyl-

3-O-methyl-α-D-manno-

pyranosyl)-(1→4)-2-O-acetyl-

6-O-benzyl-3-O-methyl-α-D-

mannopyranoside

30 87

25

Allyl 2-O-acetyl-6-O-benzyl-

4-O-tert-butyldimethylsilyl-3-O-

methyl-(α/β)-D-

mannopyranoside

20 79

26

2-O-Acetyl-6-O-benzyl-4-O-tert-

butyldimethylsilyl-3-O-methyl-

(α/β)-D-mannopyranose

21

22

23

24

26

80

81

82

84

27

1,2-di-O-Acetyl-6-O-benzyl-4-O-

tert-butyldimethylsilyl-3-O-

methyl-α-D-mannopyranose

25 82

28

1-(2-Oxopropyl)-2-O-acetyl-

6-O-benzyl-4-O-tert-

butyldimethylsilyl-3-O-methyl-

α-D-mannopyranoside

25 82

29 2-O-Acetyl-6-O-benzyl-3-O-

methyl-(α/β)-D-mannopyranose 27 84

30

(2-O-Acetyl-6-O-benzyl-3-O-

methyl-1-O-α-D-manno-

pyranosyl)-trichloroace-

timidate

28 85

62

31


methyl-1,4-O-α-D-

mannopyranosyl)-di-


28 85

32



methyl-1-O-α-D-

mannopyranosyl)-


29 86

33

2-O-Acetyl-4,6-O-benzylidene-3-

O-methyl-(α/β)-D-

mannopyranose

31 87

34

(2-O-Acetyl-4,6-O-benzylidene-

3-O-methyl-1-O-α-D-

mannopyranosyl)-


32 88

35 Methyl 3-O-methyl-α-D-

mannopyranoside

33

34 89

36 Methyl 6-O-trityl-α-D-

mannopyranoside 35 90

37 Methyl 3-O-methyl-6-O-trityl-α-

D-mannopyranoside 36 90

38 1,2,4,6-Tetra-O-acetyl-3-O-

methyl-(α/β)-D-mannopyranose 37 91

39 3-O-Methyl-(α/β)-D-

mannopyranose - -

40 4,6-O-Benzylidene-3-O-methyl-

(α/β)-D-mannopyranose - -

41

1,2-di-O-Acetyl-4,6-O-

benzylidene-3-O-methyl-(α/β)-

D-mannopyranose

- -

63

42 1,2-di-O-Acetyl-6-O-benzyl-3-O-

methyl-(α/β)-D-mannopyranose - -

43

1,2-di-O-Acetyl-6-O-benzyl-4-O-


methyl-(α/β)-D-mannopyranose

- -

64

65

4.4 Experimental Procedures

Experiment 1:

Allyl (α/β)-D-mannopyranoside 3

D-mannose (7.00 g, 0.039 mol) was dissolved in distilled allyl alcohol (46.7 mL, 0.687 mol).

Then, camphorsulfonic acid was added (46.7 mg, 0.2 mmol). The mixture was refluxed and

stirred overnight. TLC (9:1 dichloromethane-methanol) indicated that the reaction was

completed. The solvent was evaporated over vacuum until dryness was achieved, and the

mixture was concentrated. The reaction crude was applied to a column of silica gel (flash

column chromatography) which was eluted with 9:1 dichloromethane-methanol to give 3 (7.57

g, 94%, α/β > 10:1), a colourless oil.

vmax/cm-1

: 3383.79 (O-H), 1647.0 (C=C), 1060.48 (C-O)

NMR data for the α-anomer (major anomer) in accordance to those described in the literature.[23]

Experiment 2:

Allyl 4,6-O-benzylidene-(α/β)-D-mannopyranoside 4

To a solution of 3 (7.57 g, 0.037 mol) in dry THF (25 mL), benzaldehyde dimethyl acetal (11.1

mL, 0.074 mol) and camphorsulfonic acid, in a catalytic amount, were added. The mixture was

stirred and refluxed for 4 hours and 30 minutes. TLC (3:7 hexane-ethyl acetate) indicated that

the reaction was completed. The mixture was neutralized and washed with an aqueous solution

of sodium hydrogen carbonate (saturated) and extracted with ethyl acetate. The organic layer

was dried with Na2SO4, filtered and concentrated. Purification by recrystallization (9:1 hexane-

ethyl acetate) afforded 4 (6.33 g, 59 %, α/β 5:1) as a white solid (Melting point: 148 ºC).

vmax/cm-1

: 3384.58 (O-H), 1647.06 (C=C), 1094.67-1027.72 (C-O)

66

1H-NMR (CDCl3): δ 7.51-7.48 (m, Ar), 7.41-7.35 (m, Ar), 5.98-5.86 (m, -OCH2CH=CH2), 5.58

(s, -OCHPh), 5.31 (dd, 3JH-H = 17.21 Hz,

2JH-H = 1.57 Hz, -OCH2CH=CHcisHtrans), 5.23 (dd,

3JH-H

= 10.34 Hz, 2JH-H = 1.36 Hz, -OCH2CH=CHcisHtrans), 4.93 (1H, d,

3JH-H = 1.07 Hz, H-1α), 4.64

(1H, d, 3JH-H = 1.01 Hz, H-1β), 4.25, (dd,

2JH-H = 12.88 Hz,

3JH-H = 5.21 Hz, H-6a), 4.18 (dd,

2JH-H

= 12.60 Hz, 3JH-H = 4.39 Hz, -OCHaHbCH=CH2), 4.13-4.03 (m, H-2 and -OCHaHbCH=CH2),

4.03-3.77 (m, H-3,4,5,6b), 2.97 (br s, -OH).

13C-NMR (CDCl3): δ 133.51 (-OCH2CH=CH2), 129.01, 128.34 and 126.31 (Ar), 117.80

(-OCH2CH=CH2), 102.22 (-OCHPh), 99.54 (C-1), 78.86 (C-4), 71.01 (C-2), 68.67 (C-3), 68.79

and 68.26 (-OCH2CH=CH2 and C-6), 63.26 (C-5).

Experiment 3:

Allyl 4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranoside 6

To a solution of 4 (5.60 g, 0.019 mol) in dry methanol (25 mL), dibutyltin oxide (5.45 g, 0.022

mol) was added. The mixture was stirred and boiled under reflux for 3 hours. After 3 hours, the

solvent was evaporated under vacuum and the mixture was dried, using a vacuum pressure

pump. The reaction crude was dissolved in dry DMF (35 mL) and iodomethane (5.90 mL, 0.094

mol) was added. The mixture was heated at 50 ºC and stirred overnight. TLC (2:3 hexane-ethyl

acetate) indicated that the reaction was completed. The solvent was first evaporated under

vacuum until dryness was achieved. The mixture was dissolved in ethyl acetate and filtered. The

solvent of the filtrate was removed under vacuum and the reaction mixture was purified.

Purification by flash column chromatography, (eluent from 7:3 hexane-ethyl acetate to 1:1

hexane-ethyl acetate) afforded 6 (4.93 g, 80%, α/β > 10:1) as a yellowish oil.

vmax/cm-1

: 3461.67 (O-H), 1646.98 (C=C), 1093.78-1034.83 (C-O)

NMR data for the α-anomer (major anomer):

1H-NMR (CDCl3): δ 7.55-7.44 (2H, m, Ar), 7.42-7.29 (3H, m, Ar), 5.97-5.86 (1H, m,

-OCH2CH=CH2), 5.59 (1H, s, -OCHPh), 5.31 (1H, d, 3JH-H = 17.20 Hz,

2JH-H = 1.56 Hz,

-OCH2CH=CHcisHtrans), 5.23 (1H, d, 3JH-H = 10.37 Hz ,

2JH-H = 1.36 Hz -OCH2CH=CHcisHtrans),

4.94 (1H, s, 3JH-H = 1.25 Hz, H-1), 4.27 (1H, dd,

2JH-H = 8.78 Hz,

3JH-H = 3.01 Hz, H-6a), 4.21

(1H, dd, 2JH-H = 12.88 Hz,

3JH-H = 5.23 Hz, -OCHaHbCH=CH2), 4.14-4.11 (1H, m, H-2), 4.05-

67

3.98 (2H, m, H-4 and -OCHaHbCH=CH2), 3.91-3.81 (2H, m, H-5 and H-6b), 3.71 (1H, dd, 3JH-H

= 9.52, 3JH-H = 3.41 Hz, H-3), 3.56 (3H, s, -OCH3), 2.58 (1H, s, -OH).

13C-NMR (CDCl3): δ 133.47 (-OCH2CH=CH2), 129.01, 128.24 and 126.15 (Ar), 117.96

(-OCH2CH=CH2), 101.79 (-OCHPh), 99.15 (C-1), 78.73 (C-4), 77.30 (C-3), 69.18 (C-2), 68.86

and 68.25 (-OCH2CH=CH2 and C-6), 63.30 (C-5), 58.65 (-OCH3).

Experiment 4:

Allyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranoside 7

To a solution of 6 (6.00 g, 0.019 mol) in dry pyridine (40 mL) at 0 ºC, distilled acetic anhydride

(2.17 mL, 0.022 mol) and a catalytic amount of DMAP were added. The mixture was stirred at

0ºC for 5 minutes, allowed to warm room temperature and stirred for 2 hours. TLC (7:3 hexane-

ethyl acetate) indicated that the reaction was completed. The mixture was washed and

neutralized with water, and extracted with ethyl acetate. The organic layer was dried with

Na2SO4 and filtered. Ethyl acetate and pyridine were evaporated. Purification of the reaction

crude, by flash column chromatography (7:3 hexane-ethyl acetate), afforded 7 (6.20 g, 91%, α/β

> 10:1) as a colourless oil.

vmax/cm-1

: 1746.45 (C=O), 1646.97 (C=C), 1091.51-1028.98 (C-O)


1H-NMR (CDCl3): δ 7.55-7.45 (2H, m, Ar), 7.40-7.31 (3H, m, Ar), 5.97-5.85 (1H, m,

-OCH2CH=CH2), 5.61 (1H, s, -OCHPh), 5.38 (1H, dd, 3JH-H = 3.39 Hz,

3JH-H = 1.49 Hz, H-2),

5.32 (1H, dd, 3JH-H = 17.2 Hz,

2JH-H = 1.38 Hz, -OCH2CH=CHcisHtrans), 5.25 (1H, dd,

3JH-H =

10.39 Hz , 2JH-H = 0.95 Hz, -OCH2CH=CHcisHtrans), 4.84 (1H, d,

3JH-H = 1.12 Hz, H-1), 4.27 (1H,

dd, 2JH-H = 9.55 Hz,

3JH-H = 4.02 Hz, H-6a), 4.19 (1 H, dd,

2JH-H = 12.74 Hz,

3JH-H = 5.31 Hz,

-OCHaHbCH=CH2), 4.05-3.97 (2 H, m, H-4 and -OCHaHbCH=CH2), 3.90 (1H, ddd, 3JH-H =

10.12 Hz, 3JH-H = 10.12 Hz,

3JH-H = 4.37 Hz, H-5), 3.86-3.79 (2 H, m, H-3,6b), 3.46 (3H, s,

-OCH3), 2.16 (3H, s, -OCOCH3).

13C-NMR (CDCl3): δ 170.20 (-OCOCH3), 133.30 (-OCH2CH=CH2), 129.01, 128.27 and 126.19

(Ar), 118.17 (-OCH2CH=CH2), 101.86 (-OCHPh), 97.83 (C-1), 78.55 (C-4), 75.82 (C-3), 69.30

(C-2), 68.74 and 68.46 (-OCH2CH=CH2 and C-6), 63.87 (C-5), 58.42 (-OCH3), 20.99

(-OCOCH3).

68

Experiment 5:

Allyl 2-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 8

To a solution of 7 (2.74 g, 0.008 mol) in dry THF (25 mL) at 0 ºC, sodium cyanoborohydride

(5.67 g, 0.090 mol) was added. The mixture was stirred at 0 ºC, and a solution of hydrogen

chloride in dry diethyl ether 1 M (32 mL) was added portionwise (1 mL per portion) until the

reaction was completed (TLC 6:4 hexane-ethyl acetate). The mixture was evaporated under

vacuum, redissolved in water and extracted with dichloromethane. The organic layer was dried

with Na2SO4 and filtered. Purification by flash column chromatography (eluent from 7:3

hexane-ethyl acetate to 1:1 hexane-ethyl acetate) afforded 8 as a colourless oil (2.25 g, 81%, α/β

5:1).

vmax/cm-1

: 3467.07 (O-H), 1744.5 (C=O), 1646.98 (C=C), 1045.41 (C-O)

1H-NMR (CDCl3): δ 7.40-7.27 (m, Ar), 5.96-5.81 (m, -OCH2CH=CH2), 5.33-5.24 (m,

-OCH2CH=CHaHb and H-2), 5.21 (dd, 3JH-H = 10.33 Hz,

2JH-H = 1.29 Hz, -OCH2CH=CHaHb),

4.87 (1H, d, 3

JH-H = 1.56 Hz, H-1α), 4.83 (1H, d, 3JH-H = 1.58 Hz, H-1β), 4.66 (d,

2JH-H = 12.11

Hz, -OCHaHbPh) , 4.58 (d, 2JH-H = 12.11 Hz, -OCHaHbPh), 4.19 (dd,

2JH-H = 12.82 Hz,

3JH-H =

5.22 Hz, -OCHaHbCH=CH2), 4.00 (dd, 2JH-H = 12.87 Hz,

3JH-H = 6.22 Hz, -OCHaHbCH=CH2),

3.87 (dd, 3JH-H = 19.97 Hz,

3JH-H = 11.63 Hz, H-4) 3.82-3.73 (m, H-3β,5, 6a, 6b), 3.57 (1H, dd,

3JH-H = 9.47 Hz,

3JH-H = 3.02 Hz, H-3α), 3.44 (3H, s, -OCH3 β anomer), 3.42 (3H, s, -OCH3 α

anomer), 2.15 (3H, s, -OCOCH3 β anomer), 2.11 (3H, s, -OCOCH3 α anomer).

13C-NMR (CDCl3): δ 170.36 (-OCOCH3), 133.40 (-OCH2CH=CH2), 128.37, 127.64 and 127.56

(Ar), 117.94 (-OCH2CH=CH2), 97.05 (C-1α), 96.90 (C-1β), 80.00 (C-3β), 79.30 (C-3α), 73.55

(-OCH2Ph), 71.13 (C-5α), 71.06 (C-5β), 69.87 (C-6’), 68.26 (-OCH2CH=CH2), 67.56 and 67.41

(C-4 and C-2), 57.44 (-OCH3), 20.97 (-OCOCH3).

69

Experiment 6:

Allyl 2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 9

To a solution of 8 (1.80 g, 0.005 mol) in dry pyridine (15 mL) at 0 ºC, distilled acetic anhydride

(0.955 mL, 0.009 mol) and a catalytic amount of DMAP were added. The mixture was stirred at

0ºC for 5 minutes, allowed to warm room temperature and stirred for 1 hour and 30 minutes.

TLC (7:3 hexane-ethyl acetate) indicated that the reaction was completed. The mixture was

washed and neutralized with water, and extracted with ethyl acetate. The organic layer was

dried with Na2SO4 and filtered. Ethyl acetate and pyridine were evaporated. Purification of the

reaction crude, by flash column chromatography (7:3 hexane-ethyl acetate), afforded 9 (1.76 g,

88 %, α/β 8:1) as a colourless oil.

vmax/cm-1

: 1743.29 (C=O), 1647.14 (C=C), 1040.02 (C-O)


-OCH2CH=CHaHb and H-2), 5.25-5.15 (m, -OCH2CH=CHaHb and H-4), 4.88 (1H, d, 3

JH-H =

1.52 Hz, H-1α), 4.85 (1H, d, 3JH-H = 1.51 Hz, H-1β), 4.55 (2H, ABdd,

2JH-H = 11.92 Hz,

-OCH2Ph), 4.21 (dd, 2JH-H = 12.91,

3JH-H = 5.28 Hz, -OCHaHbCH=CH2), 4.02 (dd,

2JH-H = 12.8,

3JH-H = 6.2 Hz, -OCHaHbCH=CH2), 3.93 – 3.87 (m, H-5), 3.67 (dd,

3JH-H = 9.75,

3JH-H = 3.40 Hz,

H-3), 3.60 – 3.52 (m, H-6a and H-6b), 3.45 (3H, s, -OCH3 β anomer) 3.35 (3H, s, -OCH3 α

anomer), 2.15 (3H, s, -OCOCH3 β anomer), 2.13 (3H, s, -OCOCH3 α anomer), 2.05 (3H, s,

-OCOCH3 β anomer), 1.99 (3H, s, -OCOCH3 α anomer).

13C-NMR (CDCl3): δ 170.42 and 169.96 (-OCOCH3), 133.33 (-OCH2CH=CH2), 128.30, 127.76

and 127.61 (Ar), 118.07 (-OCH2CH=CH2), 96.76 (C-1α), 96.42 (C-1β), 77.04 (C-3), 73.54

(-OCH2Ph), 70.01 (C-5), 69.45 (C-6), 68.40 (OCH2CH=CH2), 68.38 (C-4), 67.87 (C-2) 57.68

(-OCH3 α anomer), 57.51 (-OCH3 β anomer), 21.01 and 20.90 (-OCOCH3).

70

Experiment 7:

2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose 10

To a solution of 9 (1.76 g, 0.004 mol) in dry methanol (15 mL), palladium (II) chloride (0.153

g, 0.860 mmol) was added. The mixture was stirred at room temperature for 2 hours. TLC (3:2

hexane-ethyl acetate) indicated that the reaction was completed. The mixture was filtered

through Celite, while washed with methanol. The filtrate was evaporated under vacuum.

Purification of the reaction crude by flash column chromatography (eluent from 7:3 hexane-

ethyl acetate to 1:1 hexane-ethyl acetate), afforded 10 (1.20 g, 75 %, α/β > 10:1) as a colourless

oil.

vmax/cm-1

: 3419.51 (O-H), 1743.65 (C=O), 1054.09 (C-O)


1H-NMR (CDCl3): δ 7.37-7.27 (5H, m, Ar), 5.34 (1H, dd,

3JH-H = 3.17,

3JH-H = 1.97 Hz, H-2),

5.24 (1H, d, 3JH-H = 1.51 Hz, H-1), 5.12 (1H, t,

3JH-H = 9.94 Hz, H-4), 4.55 (2H, s, -OCH2Ph),

4.15-4.09 (1H, m, H-5), 3.71 (1H, dd, 3JH-H = 9.73,

3JH-H = 3.32 Hz, H-3), 3.60 – 3.48 (2H, m,

H-6a and H-6b), 3.35 (3H, s, -OCH3), 2.13 (3H, s, -OCOCH3) , 2.00 (3H, s, -OCOCH3).

13C-NMR (CDCl3): δ 170.42 and 170.09 (-OCOCH3), 128.39, 128.04 and 127.80 (Ar), 92.40

(C-1), 76.48 (C-3), 73.64 (-OCH2Ph), 69.89 (C-5), 69.62 (C-6), 68.36 (C-4), 68.05 (C-2), 57.71

(-OCH3), 21.03 and 20.90 (-OCOCH3).

Experiment 8:

(2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-mannopyranosyl)-trichloroacetimidate

11

71

To a solution of 10 (1.20 g, 0.003 mol) in dry dichloromethane (15 mL) at 0ºC, distilled DBU

(0.214 mL, 1.40 mmol) and distilled trichloroacetonitrile (1.63 ml, 0.016 mol) were added

sequentially. The mixture was stirred for 10 minutes at 0ºC, allowed to warm room temperature

and stirred for 2 h. TLC (7:3 hexane-ethyl acetate) indicated that the reaction was completed.

The solvent was evaporated over vacuum. Purification of the reaction crude by flash column

chromatography (eluent from 7:3 hexane-ethyl acetate to 3:2 hexane-ethyl acetate) afforded 11

(1.27 g, 76 %) as a colourless oil.

α +38.8 (c 0.95, CH2Cl2)

vmax/cm-1

: 3316.9 (N-H), 1748.9 (C=O), 1045.4 (C-O).

1H-NMR (CDCl3): δ 8.76 (1H, s, -OC(NH)CCl3), 7.36 – 7.27 (5H, m, Ar), 6.30 (1H, d,

3JH-H =

1.89 Hz, H-1), 5.52 (1H, dd, 3JH-H = 3.20,

3JH-H = 2.14 Hz, H-2), 5.32 (1H, t,

3JH-H = 10.01 Hz,

H-4), 4.53 (2H, ABdd, 2JH-H = 11.88 Hz, -OCH2Ph), 4.11 (1H, m, H-5), 3.72 (1H, dd,

3JH-H =

9.81, 3JH-H = 3.36 Hz, H-3), 3.59 (2H, d,

3JH-H = 4.11 Hz, H-6a e H-6b), 3.38 (3H, s, -OCH3),

2.17 (3H, s, -OCOCH3), 2.00 (3H, s, -OCOCH3).

13C-NMR (CDCl3): δ 170.76, 169.98 and 168.07 (-OCOCH3 and -OC(NH)CCl3), 128.30,

127.85, 127.64 (Ar), 94.98 (C-1), 76.80 (C-3), 73.50 (-OCH2Ph), 72.73 (C-5), 69.03 (C-6),

67.66 (C-4), 66.24 (C-2), 57.92 (-OCH3), 20.90 and 20.88 (-OCOCH3).

Experiment 9:

Methyl 4,6-O-benzylidene-α-D-mannopyranoside 12

The procedure of Experiment 2 was applied to α-methyl-D-mannose (5.00 g, 0.026 mol), with

the reaction time increased to overnight. TLC (2:3 hexane-ethyl acetate) indicated that the

reaction was finished. Purification by recrystallization (9:1 hexane-ethyl acetate) afforded 12

(3.60 g, 50%) as a white solid.

IR and NMR data in accordance to those described in the literature.[24]

72

Experiment 10:

Methyl 4,6-O-benzylidene-3-O-methyl-α-D-mannopyranoside 14

The procedure of Experiment 3 was applied to compound 12 (3.60 g, 0.013 mol) with the first

step reaction time increased to overnight, and in the second step the temperature was increased

to 65ºC. TLC (2:3 hexane-ethyl acetate) indicated that the reaction was completed. Purification

of the reaction crude, by flash column chromatography (eluent from 7:3 hexane-ethyl acetate to

1:1 hexane-ethyl acetate) afforded 14 (1.75 g, 50 %) as a yellowish oil.

vmax/cm-1

: 3461.64 (O-H), 1095.18-1055.60 (C-O)

1H-NMR (CDCl3): δ 7.52-7.46 (2H, m, Ar), 7.38-7.33 (3H, m, Ar), 5.59 (1H, s, -OCHPh), 4.79

(1H, s, 3JH-H = 0.94 Hz, H-1), 4.28 (1H, dd,

2JH-H = 9.19 Hz,

3JH-H = 3.54 Hz, H-6a), 4.12-4.09

(1H, m, H-2), 4.00 (1H, t, 3JH-H = 9.28 Hz, H-4), 3.88-3.80 (2H, m, H-5 and H-6b), 3.67, (1H,

dd, 3JH-H = 7.47,

3JH-H = 2.03 Hz, H-3), 3.55 (3H, s, -OCH3), 3.40 (3H, s, -OCH3).

13C-NMR (CDCl3): δ 129.00, 128.24 and 126.17 (Ar), 101.83 (-OCHPh), 101.05 (C-1), 78.66

(C-4), 77.31 (C-3), 69.06 (C-2), 68.91 (C-6), 63.11 (C-5), 58.61 and 55.05 (-OCH3).

Experiment 11:

Methyl 2-O-acetyl-4,6-O-benzylidene-3-O-methyl-α-D-mannopyranoside 15

The procedure of Experiment 4 was applied to compound 14 (1.75 g, 5.90 mmol). TLC (7:3

hexane-ethyl acetate) indicated that the reaction was completed. Purification of the reaction

mixture by flash column chromatography (7:3 hexane-ethyl acetate) afforded 15 (1.93 g, 97 %)

as a colourless oil.

vmax/cm-1

: 3465.09 (O-H), 1747.32 (C=O), 1094.27-1060.10 (C-O)

NMR data in accordance to those described in the literature. [25]

73

Experiment 12:

Methyl 2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranoside 16

The procedure of Experiment 5 was applied to compound 15 (1.93 g, 5.70 mmol), with a

solution of hydrogen chloride in dry diethyl ether 1 M (25 mL) being added portionwise (1 mL

per portion). TLC (7:3 hexane-ethyl acetate), indicated that the reaction was completed.

Purification of the reaction mixture by flash column chromatography (eluent from 7:3 hexane-

ethyl acetate to 1:1 hexane-ethyl acetate) afforded 16 (1.94 g, 100 %) as a colourless oil.

vmax/cm-1

: 3446.2 (O-H), 1748.3-1724.8 (C=O), 1076.1 (C-O)


3JH-H = 3.08,

3JH-H = 1.83 Hz, H-2),

4.72 (1H, d, 3JH-H = 1.66 Hz, H-1), 4.66 (1H, d,

2JH-H = 11.89 Hz, -OCHaHbPh), 4.59 (1H, d,

3JH-

H = 11.90 Hz, -OCHaHbPh), 3.86 (1H, t, 3JH-H = 9.08 Hz, H-4), 3.81-3.69 (3H, m, H-5,6a,6b),

3.54 (1H, dd, 3JH-H = 9.38,

3JH-H = 3.21 Hz, H-3), 3.40 (3H, s, -OCH3), 3.39 (3H, s, -OCH3), 2.13

(3H, s, -OCOCH3).

13C-NMR (CDCl3): δ 170.41 (-OCOCH3), 128.60, 128.10 and 127.90 (Ar), 98.98 (C-1), 78.92

(C-3), 73.87 (-OCH2Ph), 70.21 (C-6), 70.11 (C-5), 68.25 (C-4), 67.02 (C-2), 57.04 and 55.29

(-OCH3), 20.90 (-OCOCH3).

Experiment 13:

Methyl (2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-2-O-acetyl-

6-O-benzyl-3-O-methyl-α-D-mannopyranoside 17

To a solution of 11 (0.200 g, 0.391 mmol) and 16 (0.133 g, 0.391 mmol) in dry dichloromethane

(4 mL), finely powdered molecular sieves (4 Å) were first added. The solution was stirred for 30

minutes at room temperature. At -20 ºC, distilled TMSOTf (71 μL, 0.391 mmol) was added and

74

the mixture was stirred at this temperature for another 30 minutes. TLC (3:2 hexane-ethyl

acetate) indicated that the reaction was finished. The mixture was neutralized and washed with

an aqueous solution of sodium hydrogen carbonate (saturated) and extracted with

dichloromethane. The organic layer was dried with Na2SO4, filtered and concentrated.

Purification by silica preparative TLC (3:2 hexane-ethyl acetate) afforded 17 (0.187 g, 69 %) as

a colourless oil.

α +50.4 (c 1.04, CH2Cl2)

vmax/cm-1

: 1746.15 (C=O), 1044.43 (C-O)


3JH-H = 2.94 Hz,

3JH-H = 2.15 Hz, H-

2A), 5.29 (1H, dd, 3

JH-H = 3.21 Hz, 3JH-H = 1.85 Hz, H-2B), 5.23 (1H, d,

3JH-H = 1.75 Hz, H-1A),

5.17 (1H, t, 3

JH-H = 9.96 Hz, H-4A), 4.71 (1H, d, 3JH-H = 1.62 Hz, H-1B), 4.61-4.40 (4H, m, -

OCH2PhA and OCH2PhB), 3.91-3.83 (2H, m, H-4B,5A), 3.83-3.71 (3H, m, H-5B,6B,6’B), 3.63

(1H, dd, 3JH-H = 9.09,

3JH-H = 3.30 Hz, H-3B), 3.56 (1H, dd,

3JH-H = 9.77,

3JH-H = 3.23 Hz, H-3A),

3.48-3.40 (5H, m, H-6A, H-6’A and -OCH3), 3.39 (3H, s, -OCH3), 3.34 (3H, s, -OCH3), 2.10

(3H, s, -OCOCH3), 2.08 (3H, s, -OCOCH3), 1.98 (3H, s, -OCOCH3).

13C-NMR (CDCl3): δ 170.31, 170.11 and 169.90 (-OCOCH3), 128.27, 127.81 and 127.38 (Ar),

99.46 (C-1A), 98.57 (C-1B), 79.95 (C-3B), 76.86 (C-3A), 73.99, 70.85 and 70.73 (C-4B,C-5A and

C-5B), 73.53 and 73.29 (-OCH2PhA and -OCH2PhB), 69.84 and 69.45 (C-6A and C-6B), 68.25

(C-4A), 67.88 and 67.42 (C-2A and C-2B), 57.56, 57.18 and 55.13 (-OCH3), 21.01, 20.96 and

20.92 (-OCOCH3).

Experiment 14:

Methyl (6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-6-O-benzyl-3-O-methyl-α-D-

mannopyranoside 18

To a solution of 17 (0.245 g, 0.355 mmol) in dry methanol (1 mL), sodium methoxide (0.023 g,

0.426 mmol) was added. The mixture was stirred for 1 hour and 30 minutes at room

temperature. TLC (1:4 hexane-ethyl acetate) indicated that the reaction was not completed.

More quantity of sodium methoxide was added (0.011 g, 0.213 mmol) and after 1 h the reaction

was completed. The mixture was washed with an aqueous solution of ammonium chloride

75

(saturated) and extracted with ethyl acetate. The organic layer was dried with Na2SO4, filtered

and concentrated. Purification by silica preparative TLC (100 % ethyl acetate) afforded 18

(0.196 g, 98 %) as a colourless viscous foam.

α +55.4 (c 0.95, CH2Cl2)

vmax/cm-1

: 3443.65 (O-H), 1043.25 (C-O)

1H-NMR (CDCl3): δ 7.35-7.27 (10 H, m, Ar), 5.30 (1 H, d,

3JH-H = 1.74 Hz, H-1), 4.81 (1 H, d,

3JH-H = 1.61 Hz, H-1), 4.61-4.43 (4H, m, -OCH2PhA and OCH2PhB), 4.07-4.03 (2H, m, H-2,5),

3.93 (1H, t, 3JH-H = 9.12 Hz, H-4), 3.86 (1H, t,

3JH-H = 9.35 Hz, H-4), 3.77-3.71 (4H, m, H-

2,5,6,6’), 3.63 (1H, dd, 2JH-H = 10.01 Hz,

3JH-H = 4.56 Hz, H-6), 3.58 (1H, dd,

2JH-H = 9.99 Hz,

3JH-H = 4.90 Hz, H-6’), 3.53 (1H, dd,

3JH-H = 8.97 Hz,

3JH-H = 3.34 Hz, H-3), 3.49 (3H, s,

-OCH3), 3.43 (3H, s, -OCH3), 3.40 (3H, s, -OCH3), 3.37 (1H, dd, 3JH-H = 9.10 Hz,

3JH-H = 3.17

Hz, H-3).

13C-NMR (CDCl3): δ 128.40, 128.35, 127.75 (Ar), 100.47 and 99.03 (C-1A and C-1B), 80.37

and 77.87 (C-3A and C-3B), 73.43 and 73.34 (-OCH2PhA and -OCH2PhB), 73.23 (C-4), 71.25

and 70.19 (C-2 and C-5), 69.96 and 69.63 (C-6A and C-6B), 67.55 (C-4), 67.13 and 66.87 (C-2

and C-5), 57.59, 57.25 and 55.24 (-OCH3).

Experiment 15:

Methyl (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-D-mannopyranoside 1

Compound 18 (0.295 g, 0,523 mmol) in ethyl acetate /ethanol 1:1 (6 mL), was hydrogenated

overnight at 50 psi in the presence of Pd/C 10% (0.200 g). The mixture was filtered through

Celite, while washed with methanol and water. The filtrate was evaporated under vacuum,

which afforded 1 (0.201 g, 100%) as a colourless viscous foam.

α +67.5 (c 0.99, H2O)

1H-NMR (D2O): δ 5.13 (1H, d,

3JH-H = 1.80 Hz, H-1), 4.72 (1H, d,

3JH-H = 1.73 Hz, H-1), 4.13

(1H, dd, 3JH-H = 2.86 Hz,

3JH-H = 2.17 Hz, H-2), 4.09 (1H, dd,

3JH-H = 3.12 Hz,

3JH-H = 1.98 Hz,

H-2), 3.83-3.58 (8H, m, H-4A,4B,5A,5B, 6A,6’A, 6B,6’B), 3.53 (dd, 3JH-H = 9.09 Hz,

3JH-H = 3.26

76

Hz, H-3), 3.40 (1H, dd, 3JH-H = 9.13 Hz,

3JH-H = 3.09 Hz, H-3) 3.38-3.35 (6H, m, -OCH3), 3.33

(3H, s, -OCH3).

13C-NMR (D2O): δ 101.39 and 100.66 (C-1A and C-1B), 80.99 and 79.72 (C-3A and C-3B),

73.73, 72.65, 70.99 and 65.45 (C-4A, C-4B , C-5A, and C-5B), 66.03 and 65.66 (C-2A and C-2B),

60.95 and 60.86 (C-6A and C-6B), 56.22, 56.12 and 54.81 (-OCH3).

Experiment 16:

Allyl (2,4-di-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-2-O-acetyl-6-

O-benzyl-3-O-methyl-(α/β)-D-mannopyranoside 19

The glycosylation reaction of donor 11 (0.699 g, 1.40 mmol) and acceptor 8 (0.500 g, 1.40

mmol) was performed according to the procedure described in Experiment 13. TLC (3:2

hexane-ethyl acetate) indicated that the reaction was completed. Purification by flash column

chromatography (3:2 hexane-ethyl acetate) afforded 19 (0.750 g, 77 %, αα/ αβ 9:1) as a

colourless oil.

α +35.1 (c 1.05, CH2Cl2)

vmax/cm-1

: 1746.63 (C=O), 1652.68 (C=C), 1047.17 (C-O)

1H-NMR (CDCl3): δ 7.35-7.28 (m, Ar), 5.98-5.87 (m, -OCH2CH=CH2), 5.39-5.37 (m, H-2A),

5.34-5.28 (m, -OCH2CH=CHaHb and H-2B), 5.25-5.21 (m, -OCH2CH=CHaHb and H-1A), 5.17

(t, 3

JH-H = 10.01 Hz, H-4A), 4.86 (1H, d, 3JH-H = 1.21 Hz, H-1Bα), 4.83 (1H, d,

3JH-H = 1.39 Hz,

H-1Bβ), 4.60-4.41 (m, -OCH2PhA and OCH2PhB), 4.21 (dd, 2JH-H = 12.80,

3JH-H = 5.30 Hz,

-OCHaHbCH=CH2), 4.02 (dd, 2JH-H = 13.02,

3JH-H = 6.19 Hz, -OCHaHbCH=CH2), 3.92 – 3.77

(m, H-4B,5A,5B,6B), 3.74 (dd, 3JH-H = 11.10 Hz,

3JH-H = 5.52 Hz, H-6’B), 3.67 (dd,

3JH-H = 8.85,

3JH-H = 3.32 Hz, H-3B), 3.57 (dd,

3JH-H = 9.77,

3JH-H = 3.12 Hz, H-3A), 3.49-3.39 (m, H-6A, H-6’A

and -OCH3), 3.34 (s, -OCH3), 2.10 (s, -OCOCH3), 2.09 (s, -OCOCH3), 1.98 (s, -OCOCH3).

13C-NMR (CDCl3): δ 170.28, 170.10 and 168.04 (-OCOCH3), 133.44 (-OCH2CH=CH2),

128.23, 127.81 and 127.36 (Ar), 118.04 (-OCH2CH=CH2), 99.50 (C-1A), 96.72 (C-1B), 79.96

(C-3B), 76.84 (C-3A), 74.04, 70.85 and 70.92 (C-4B, 5A, 5B), 73.53 and 73.26 (-OCH2PhA and

77

OCH2PhB), 69.83 and 69.47 (C-6A and C-6B), 68.44 (OCH2CH=CH2), 68.28 (C-4A), 67.69 and

67.56 (C-2A and C-2B), 57.57 and 57.19 (-OCH3), 21.01, 20.95 and 20.92 (-OCOCH3).

Experiment 17:

(2,4-di-O-Acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-2-O-acetyl-6-O-

benzyl-3-O-methyl-(α/β)-D-mannopyranose 20


hexane-ethyl acetate) indicated that the reaction was completed. Purification by flash column

chromatography (eluent from 7:3 hexane-ethyl acetate to 1:1 hexane-ethyl acetate) afforded 20

(0.570 g, 80 %, αα/ αβ > 10:1) as a colourless viscous foam.

α +39.8 (c 0.98, CH2Cl2)

vmax/cm-1

: 3418.6 (O-H),1743.5 (C=O), 1108.85-1044.3 (C-O)

NMR data for the αα-anomer (major anomer):


3JH-H= 3.06 Hz,

3JH-H= 2.06 Hz, H-

2A), 5.32 (1H, 3JH-H= 3.09 Hz,

3JH-H= 1.96 Hz, H-2B), 5.22-5.21 (2H, m, H-1A and H-1B), 5.16

(1H, t, 3

JH-H = 9.96 Hz, H-4A), 4.57-4.42 (4H, m, -OCH2PhA and OCH2PhB), 4.09-4.03 (1H, m,

H-5B), 3.85-3.78 (3H, m, H-4B, 5A,6B), 3.73 – 3.66 (2H, m, H-3B,6’B), 3.55 (1H, dd, 3JH-H = 9.70

Hz, 3JH-H = 3.25 Hz, H-3A), 3.44 (1H, dd,

2JH-H = 10.58 Hz,

3JH-H = 5.41 Hz, H-6A), 3.40 (3H, s,

-OCH3), 3.38-3.33 (4H, m, H-6’ A and -OCH3), 2.10 (3H, s, -OCOCH3), 2.09 (3H, s,

-OCOCH3), 2.00 (3H, s, -OCOCH3).

13C-NMR (CDCl3): δ 170.31, 170.11 and 169.92 (-OCOCH3), 128.28, 127.82 and 127.59 (Ar),

99.59 and 92.06 (C-1A and C-1B), 79.50 (C-3B), 76.85 (C-3A), 74.49 and 70.12 (C-4B and C-5A),

73.53 and 73.22 (-OCH2PhA and -OCH2PhB), 70.92 (C-5B), 70.09 (C-6B), 69.32 (C-6A), 68.16

(C-4A), 67.67 (C-2A and C-2B), 57.55 and 57.16 (-OCH3), 20.96 and 20.90 (-OCOCH3).

78

Experiment 18:

(6-O-Benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-6-O-benzyl-3-O-methyl-(α/β)-D-

mannopyranose 21

To a solution of 20 (0.470 g, 0.069 mmol) in dry methanol (2 mL), sodium methoxide (0.068 g,

1.25 mmol) was added. The mixture was stirred for 2 hours and 30 minutes at room

temperature. TLC (100 % ethyl acetate) indicated that the reaction was not completed. More

quantity of sodium methoxide was added (0.011 g, 0.213 mmol) and the mixture was stirred for

more 4 hours. TLC with the same eluent indicated the reaction was completed. The mixture was

diluted with methanol and Dowex-H+

resin was added until neutral pH. The mixture was filtered

and the filtrate concentrated. Purification by flash column chromatography (100 % ethyl

acetate) of the filtrate afforded 21 (0.297 g, 78 %, αα/ αβ > 10:1) as a colourless viscous foam.

α +51.8 (c 0.95, CH2Cl2)

vmax/cm-1

: 3418.64 (O-H), 1101.36-1049.2 (C-O)

NMR data for the αα-anomer (major anomer):

1H-NMR (CDCl3): δ 7.36-7.27 (10 H, m, Ar), 5.29-5.21 (2 H, m, H-1A and H-1B), 4.60-4.43

(4H, m, -OCH2PhA and OCH2PhB), 4.05 (1H, br s, H-2), 4.03-3.52 (10H, m, H-2,3,4A,4B,5A,5B,

6A,6’A, 6B,6’B), 3.48 (3H, s, -OCH3), 3.41 (3H, s, -OCH3), 3.35 (1H, dd, 3JH-H = 9.18 Hz,

3JH-H =

2.95 Hz, H-3).

13C-NMR (CDCl3): δ 128.43, 128.37, 127.76 (Ar), 101.17 and 93.79 (C-1A and C-1B), 81.38

and 80.68 (C-3A and C-3B), 73.63 and 73.41 (-OCH2PhA and -OCH2PhB), 73.35 (C-4), 71.32

and 70.19 (C-2 and C-5), 70.28 and 69.90 (C-6A and C-6B), 67.73 (C-4), 67.06 and 66.99 (C-2

and C-5), 57.20 and 56.62 (-OCH3).

79

Experiment 19:

(3-O-Methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-(α/β)-D-mannopyranose 2

Compound 21 (0.280 g, 0.509 mmol) in ethyl acetate/ethanol 5:1 (6 mL), was hydrogenated for

7 hours at 50 psi in the presence of Pd/C 10% (0.100 g). The mixture was filtered through

Celite, while washed with methanol and water. The filtrate was evaporated under vacuum

affording 2 (0.181 g, 98%, αα/ αβ 2:1) as a colourless viscous foam.

α +57.4 (c 0.96, MeOH)

vmax/cm-1

: 3335.4 (O-H), 1041.2 (C-O)

1H-NMR (D2O): δ 5.18-5.11 (m, H-1A and H-1Bα), 4.81 (br s, H-1Bβ), 4.16 (br s, H-2), 4.12 (br

s, H-2 Bβ), 4.08 (br s, H-2), 3.86-3.55 (m, H-3,4A,4B,5A,5B, 6A,6’A, 6B,6’B), 3.51-3.45 (m, H-3 Bβ),

3.44-3.37 (m, H-3, -OCH3A and –OCH3B).

13C-NMR (D2O): δ 101.36 and 93.78 (C-1A and C-1Bα), 93.66 (C-1Bβ), 83.21 (C-3Bβ), 80.70 and

79.69 (C-3A and C-3Bα), 73.70, 72.71, 70.91 and 65.45 (C-4A, C-4B , C-5A, and C-5B), 66.44 and

66.03 (C-2A and C-2B), 60.98 and 60.86 (C-6A and C-6B),56.18 and 56.08 (-OCH3).

Experiment 20:

Allyl 2-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-(α/β)-D-mannopyrano-

side 25

To a solution of 8 (0.475 g, 1.29 mmol) in dry dichloromethane (3 mL) at 0ºC, dry DIPEA

(0.633 mL, 3.63 mmol) and TBDMSOTf (0.596 mL, 2.60 mmol) were added sequentially. The

mixture was stirred for 20 minutes at 0ºC. TLC (4:1 hexane-ethyl acetate) indicated that the

reaction was completed. The mixture was washed with an aqueous solution of sodium hydrogen

carbonate (saturated) and extracted with dichloromethane. The organic layer was dried with

80

Na2SO4, filtered and concentrated. Purification of the reaction crude by flash column

chromatography (eluent from 100 % hexane to 4:1 hexane-ethyl acetate) afforded 25 (0.560 g,

89 %, α/β 5:1) as a colourless oil.

vmax/cm-1

:1747.83 (C=O), 1647.34 (C=C), 1107.61-1058.19 (C-O)


-OCH2CH=CHaHb and H-2), 5.21 (dd, 3JH-H = 10.42 Hz,

2JH-H = 1.50 Hz, -OCH2CH=CHaHb),

4.86 (1H, d, 3

JH-H = 1.59 Hz, H-1α), 4.82 (1H, d, 3JH-H = 1.69 Hz, H-1β), 4.60 (ABdd,

2JH-H =

12.14 Hz, -OCH2Ph) , 4.21 (dd, 2JH-H = 12.99 Hz,

3JH-H = 5.23 Hz, -OCHaHbCH=CH2), 4.01 (dd,

2JH-H = 12.93 Hz,

3JH-H = 6.27 Hz, -OCHaHbCH=CH2), 3.88-3.66 (m, H-4,5,6a,6b), 3.44-3.40 (m,

H-3), 3.32 (3H, s, -OCH3 β anomer), 3.30 (3H, s, -OCH3 α anomer), 2.11 (3H, s, -OCOCH3 β

anomer), 2.10 (3H, s, -OCOCH3 α anomer), 0.92 (s, tert-butyl), 0.83 (s, tert-butyl), 0.06 (s,

Si-CH3) and 0.01 (s, Si-CH3).

13C-NMR (CDCl3): δ 170.44 (-OCOCH3), 133.64 (-OCH2CH=CH2), 128.26 and 127.43 (Ar),

117.80 (-OCH2CH=CH2), 96.84 (C-1α), 96.61 (C-1β), 79.85 (C-3), 73.25 (-OCH2Ph), 72.77

(C-5), 69.60 (C-6a and C-6b), 68.21 (-OCH2CH=CH2), 67.88 and 67.71 (C-4 and C-2), 56.75

(-OCH3), 25.98, 25.89 (tert-butyl), 21.02 (-OCOCH3), -4.05 and -5.16 (Si-CH3).

Experiment 21:

Attempted synthesis of 2-O-Acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-

(α/β)-D-mannopyranose 26

A solution of bis(dibenzylideneacetone)palladium (0) (5.98 mg, 0.01 mmol) and 1,4-

Bis(diphenylphosphino)butane (0.044 g, 0.104 mmol) in dry THF (1 mL) was stirred at room

temperature for 15 minutes. This mixture was then added to a stirred solution of 25 (0.050 g,

0.104 mmol) in dry THF, followed by addition of 1,3-dimethylbarbituric acid (0.032 g, 0.208

mmol). The solution was stirred at the same temperature for 30 minutes. TLC (4:1 hexane-ethyl

acetate) indicated that the reaction did not occur. The mixture was stirred at 60 ºC for 30

minutes. After 30 minutes TLC with the same eluent indicated again that the reaction did not

occur. The solution was stirred overnight at this temperature. TLC indicated that the starting

material was not consumed.

81

Experiment 22:



To a solution of 25 (0.050 g, 0.104 mmol) in dry THF at 0ºC, sodium borohydride (5.12 mg,

0.135 mmol) and iodine (1.32 mg, 0.005 mmol) were added sequentially. The mixture was

stirred at 0ºC for 20 minutes. TLC (1:4 hexane-ethyl acetate) indicated that the reaction did not

occur. The mixture was stirred for more 3 hours. TLC with the same eluent indicated that the

starting material was not consumed.

Experiment 23:



To a solution of 25 (0.025 g, 0.052 mmol) in dry DMF (1 mL) at 60 ºC, t-BuOK (0.012 g, 0.104

mmol) was added. The solution was stirred at this temperature for 1 hour. TLC (4:1 hexane-

ethyl acetate) indicated that the initial product was totally consumed. Interpretation of the 1H-

NMR spectrum of the reaction mixture revealed that the obtained product was not the expected

compound. A partially deprotected product was obtained.

82

Experiment 24:



To a solution of 25 (0.075 g, 0.156 mmol) in distilled ethyl acetate, an aqueous solution of

acetic acid (90 % v/v), sodium acetate (0.077 g, 0.936 mmol) and palladium (II) chloride (0.041

g, 0.234 mmol) were added sequentially. The mixture was stirred overnight at room

temperature. TLC (4:1 hexane-ethyl acetate) revealed that the initial product was totally

consumed. The reaction mixture was filtered through Celite, while washed with ethyl acetate.

The filtrate was washed with an aqueous solution of sodium hydrogen carbonate (saturated) and

extracted twice with dichloromethane. The combined organic layers were dried over Na2SO4

and concentrated. Purification by silica preparative TLC (7:3 hexane-ethyl acetate), afforded

0.053 g of a mixture of products.

Experiment 25:

1,2-di-O-Acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-α-D-mannopyranose

27

1-(2-Oxopropyl)-2-O-acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-α-D-

mannopyranoside 28

The procedure of Experiment 6 was applied to the mixture of products afforded by Experiment

24 (0.053 mg, 0.120 mmol) with the reaction time increased to overnight. TLC (4:1 hexane-

ethyl acetate) indicated that the reaction occurred in only one of the compounds. After the work-

83

up procedure, purification by silica preparative TLC (7:3 hexane-ethyl acetate) afforded product

27 (0.026 g, 35 %, 2 steps) and compound 28 (0.020 g, 29 %, 1 step) as colourless oils.

Compound 27

vmax/cm-1

:1749.50 (C=O),1254.64 (C-Si), 1108.81-1025.71 (C-O)

NMR data:

1H-NMR (CDCl3): δ 7.29-7.19 (5H, m, Ar), 6.03 (1H, d,

3JH-H = 1.90 Hz, H-1), 5.24 (1H, dd,

3JH-H = 3.18 Hz,

3JH-H = 2.13 Hz, H-2), 4.59 (1H, ABdd,

2JH-H = 12.12 Hz, -OCH2Ph), 3.81 (1H,

t, 3JH-H = 9.31 Hz, H-4), 3.71 (1H, ddd,

3JH-H = 9.50 Hz,

3JH-H = 4.35 Hz,

3JH-H = 2.43 Hz, H-5),

3.67- 3.62 (2H, m, H-6a,6b), 3.33 (1H, dd, 3JH-H = 9.04 Hz,

3JH-H = 3.33 Hz, H-3), 3.25 (3H, s,

-OCH3), 2.06 (3H, s, -OCOCH3), 2.04 (3H, s, -OCOCH3), 0.76 (9H, tert-butyl), 0.07 (3H, s,

Si-CH3) and 0.01 (3H, s, Si-CH3).


(C-3), 75.34 (C-5), 73.38 (-OCH2Ph), 69.07 (C-6a and C-6b), 67.20 (C-4) , 66.49 (C-2), 56.97

(-OCH3), 25.95 (tert-butyl), 21.03 and 20.86 (-OCOCH3), -4.10, -5.22 (Si-CH3).

Compound 28

vmax/cm-1

:1746.89 (C=O),1257.79 (C-Si), 1091.94 (C-O)

NMR data:


3JH-H = 3.25 Hz,

3JH-H = 1.81 Hz, H-

2), 4.86 (1H, d, 3

JH-H = 1.60 Hz, H-1), 4.58 (2H, ABdd, 2JH-H = 12.10 Hz, -OCH2Ph), 4.24 (1H,

d, 2JH-H = 17.27 Hz, -OCHaHbCOCH3), 4.13 (1H, d,

2JH-H = 17.26 Hz, -OCHaHbCOCH3),

3.81-3.62 (4H, m, H-4,5,6a,6b), 3.45 (1H, dd, 3JH-H = 8.52 Hz,

3JH-H = 3.33 Hz, H-3), 3.32 (3H, s,

-OCH3), 2.15 (3H, s, -OCOCH3 or -OCH2COCH3), 2.10 (3H, s, -OCOCH3 or -OCH2COCH3),

0.84 (9H, tert-butyl), 0.08 (3H, s, Si-CH3) and 0.02 (3H, s, Si-CH3).

13C-NMR (CDCl3): δ 204.79 (-OCH2COCH3), 170.23 (-OCOCH3), 128.30, 127.50 and 127.46

(Ar), 97.69 (C-1), 79.70 (C-3), 73.30 (-OCH2Ph), 73.26 (C-5), 71.77 (-OCH2COCH3), 69.47

(C-6a and C-6b), 67.70 and 67.34 (C-4 and C-2), 56.89 (-OCH3), 25.94 (tert-butyl), 26.46 and

20.95 (-OCOCH3 and -OCH2COCH3), -4.07, -5.20 (Si-CH3).

84

Experiment 26:



To a solution of 25 (0.100 g, 0.208 mmol) in dry THF at 0 ºC, (dimethyl sulfide)trihydroboron

(52 μL, 0.104 mmol) was added. The mixture was stirred at this temperature for 20 minutes.

TLC (7:3 hexane-ethyl acetate) indicated that the inicial product was totally consumed and the

formation of several products. The crude was purified by silica preparative TLC (7:3 hexane-

ethyl acetate). Interpretation of the 1H-NMR spectrum from the different products revealed that

none of them was the expected compound.

Experiment 27:

2-O-Acetyl-6-O-benzyl-3-O-methyl-(α/β)-D-mannopyranose 29


hexane-ethyl acetate) indicated that the reaction was completed. Purification by silica

preparative TLC (1:1 hexane-ethyl acetate) afforded 29 (0.102 g, 73 %, α/β > 10:1) as a

colourless oil.

vmax/cm-1

: 3445.38 (O-H), 1747.76 (C=O), 1075.53 (C-O)



3JH-H = 3.08 Hz,

3JH-H = 1.81 Hz,

H-2), 5.18 (1H, d, 3

JH-H = 1.43 Hz, H-1), 4.58 (2H, ddAB, 2JH-H = 12.04 Hz, -OCH2Ph), 4.05

(1H, ddd, 3JH-H =9.62 Hz,

3JH-H =6.90 Hz,

3JH-H = 2.65 Hz, H-5), 3.79 (1H, dd,

2JH-H =10.32 Hz,

3JH-H = 2.70 Hz, H-6a), 3.72 (1H, t,

3JH-H = 9.61 Hz, H-4), 3.68 (1H, dd,

2JH-H =10.57 Hz,

3JH-H =

3.70 Hz, H-6b), 3.58 (1H, dd, 3JH-H = 9.49 Hz,

3JH-H = 3.23 Hz, H-3), 3.40 (3H, s, -OCH3), 2.10

(3H, s, -OCOCH3).

85


(C-3), 73.57 (-OCH2Ph), 71.02 (C-5), 70.14 (C-6a and C-6b), 67.71 and 67.46 (C-4 and C-2),

57.38 (-OCH3), 20.95 (-OCOCH3).

Experiment 28:

(2-O-Acetyl-6-O-benzyl-3-O-methyl-1-O-α-D-mannopyranosyl)-trichloroacetimidate 30

(2-O-Acetyl-6-O-benzyl-3-O-methyl-1,4-O-α-D-mannopyranosyl)-di-trichloroacetimidate

31

The procedure of Experiment 8 was applied to compound 29 (0.042 g, 0.129 mol) using

different equivalents of distilled DBU (1.9 μL, 0.013 mmol) and distilled trichloroacetonitrile

(26 μL, 0.257 mmol). TLC (7:3 hexane-ethyl acetate) indicated the formation of two different

compounds. Purification of the reaction crude, by silica preparative TLC (7:3 hexane-ethyl

acetate) afforded 30 (0.022 g, 36 %) and 31 (0.047 g, 59 %) as colourless oils.

Compound 30


3JH-H =

1.58 Hz, H-1), 5.48 (1H, dd, 3JH-H = 3.17 Hz,

3JH-H = 2.12 Hz, H-2), 4.65 (1H, d,

2JH-H = 12.00

Hz, -OCH2Ph), 4.57 (1H, d, 2JH-H = 11.97 Hz, -OCH2Ph), 4.06-3.97 (2H, m, H-4,6a), 3.85-3.74

(2H, m, H-5,6b), 3.63 (1H, dd, 3JH-H = 8.77 Hz,

3JH-H = 3.02 Hz, H-3), 3.45 (3H, s, -OCH3), 2.15

(3H, s, -OCOCH3).

13C-NMR (CDCl3): δ 169.99 and 168.32 (-OCOCH3 and -OC(NH)CCl3), 128.40, 127.72 and

127.65 (Ar), 95.35 (C-1), 79.11 (C-3), 73.78 (-OCH2Ph), 73.60 (C-4), 69.51 (C-5), 67.10 (C-6),

65.85 (C-2), 57.78 (-OCH3), 20.84 (-OCOCH3).

86

Compound 31

1H-NMR (CDCl3): δ 8.78 (1H, s, -OC(NH)CCl3), 8.55 (1H, s, -OC(NH)CCl3), 7.35 – 7.27 (5H,

m, Ar), 6.35 (1H, d, 3JH-H = 1.72 Hz, H-1), 5.62 (1H, t,

3JH-H = 10.02 Hz, H-4), 5.56 (1H, dd,

3JH-H = 3.29 Hz,

3JH-H = 2.04 Hz, H-2), 4.56 (2H, ABdd,

2JH-H = 12.40 Hz, -OCH2Ph), 4.22 (1H,

ddd, 3JH-H =10.22 Hz,

3JH-H = 3.68 Hz,

3JH-H =3.68 Hz, H-5), 3.84 (1H, dd,

3JH-H = 9.79 Hz,

3JH-H

= 3.39 Hz, H-3), 3.67 (2H, d, 3JH-H = 3.73 Hz, H-6a e H-6b), 3.41 (3H, s, -OCH3), 2.18 (3H, s,

-OCOCH3).

13C-NMR (CDCl3): δ 170.55, 168.33 and 168.12 (-OCOCH3 and -OC(NH)CCl3), 128.27,

127.79 and 127.60 (Ar), 95.10 (C-1), 77.86 (C-3), 73.43 (-OCH2Ph), 73.14 (C-5), 71.58 (C-4),

68.27 (C-6), 66.51 (C-2), 58.21(-OCH3), 20.92 (-OCOCH3).

Experiment 29:

(2-O-Acetyl-6-O-benzyl-4-O-tert-butyldimethylsilyl-3-O-methyl-1-O-α-D-mannopyra-

nosyl)-trichloroacetimidate 32


hexane-ethyl acetate) indicated that the reaction was completed. Purification by silica

preparative TLC (4:1 hexane-ethyl acetate) afforded 32 (0.020 g, 74 %) as a colourless oil.


3JH-H =

1.49 Hz, H-1), 5.50 (1H, dd, 3JH-H = 3.21 Hz,

3JH-H = 2.32 Hz, H-2), 4.59 (1H, s, -OCH2Ph),

3.99-3.89 (2H, m, H-5,6a), 3.79-3.71 (2H, m, H-4,6b), 3.47 (1H, dd, 3JH-H = 8.36 Hz,

3JH-H =

3.22 Hz, H-3), 3.33 (3H, s, -OCH3), 2.13 (3H, s, -OCOCH3), 0.84 (9H, tert-butyl), 0.08 (3H, s,

Si-CH3) and 0.03 (3H, s, Si-CH3).

13C-NMR (CDCl3): δ 170.40 and 167.89 (-OCOCH3 and -OC(NH)CCl3), 128.37, 127.92 and

127.69 (Ar), 97.70 (C-1), 79.30 (C-3), 73.32 (-OCH2Ph), 72.24 (C-5), 69.93 (C-6), 68.28 and

68.06 (C-2 and C-4), 56.79 (-OCH3), 25.92 (tert-butyl), 21.03 (-OCOCH3), -4.06, -5.19

(Si-CH3).

87

Experiment 30:

Methyl (2-O-acetyl-6-O-benzyl-3-O-methyl-α-D-mannopyranosyl)-(1→4)-2-O-acetyl-6-O-

benzyl-3-O-methyl-α-D-mannopyranoside 24

The glycosylation reaction of donor 31 (0.047 g, 0.076 mmol) and acceptor 16 (0.026 g, 0.076

mmol) was performed according to the procedure described in Experiment 13, with the reaction

time increased to overnight. TLC (1:1 hexane-ethyl acetate) indicated that the reaction was

completed. Purification by silica preparative TLC (1:1 hexane-ethyl acetate) afforded 24 (0.011

g, 18 %, mostly the α anomer) as a colourless viscous foam.

vmax/cm-1

: 3420.22 (O-H), 1739.85 (C=O), 1072.58 (C-O)

NMR data for the α anomer:

1H-NMR (CDCl3): δ 7.39-7.27 (10 H, m, Ar), 5.38 (1H, br s, H-2), 5.34-5.31 (1H, m, H-2), 5.28

(1H, br s, H-1), 5.23 (1H, br s, H-1), 4.66-4.53 (4H, m, -OCH2PhA and OCH2PhB), 3.85-3.67

(m, H-3),3.63-3.56 (4H, m, H-3 and -OCH3), 3.42 (3H,s, -OCH3), 3.36 (3H, s, -OCH3), 2.15

(3H, s, -OCOCH3), 2.12 (3H, s, -OCOCH3).

13C-NMR (CDCl3): δ 128.46, 128.42 and 128.06 (Ar), 92.79 and 92.55 (C-1’A and C-1’B),

80.91 and 78.87 (C-3A and C-3B), 73.72 and 73.67 (-OCH2PhA and -OCH2PhB), 71.16; 70.14

and 68.47 (C-6A and C-6B), 69.15, 68.44; 67.97 and 67.54 (C-2A and C-2B),67.45; 57.88, 57.85

and 57.41 (-OCH3), 21.67 and 20.97 (-OCOCH3).

Experiment 31:

2-O-Acetyl-4,6-O-benzylidene-3-O-methyl-(α/β)-D-mannopyranose 33

The procedure of Experiment 24 was applied to compound 7 (0.100 g, 0.274 mmol), with the

reaction time decreased to 5 hours. TLC (1:1 hexane-ethyl acetate) indicated that the reaction

88

was completed. Purification by silica preparative TLC (1:1 hexane-ethyl acetate) afforded 33

(0.070 g, 78 %, α/β 6:1) as a colourless oil.

vmax/cm-1

: 3382.32 (O-H), 1748.45 (C=O), 1101.41-1056.28 (C-O)

1H-NMR (CDCl3): δ 7.53-7.46 (m, Ar), 7.39-7.33 (m, Ar), 5.61 (s, -OCHPh), 5.36 (dd,

3JH-H =

3.25 Hz, 3JH-H = 1.42 Hz, H-2), 5.16 (d,

3JH-H = 1.12 Hz, H-1α), 5.11 (d,

3JH-H = 1.11 Hz, H-1β),

4.27-4.21 (m, H-6a), 4.17-4.05 (m, H-5), 3.99 (t, 3JH-H = 9.58 Hz, H-4), 3.88-3.78 (m, H-3,6b),

3.48 (3H, s, -OCH3 β anomer), 3.46 (3H, s, -OCH3 α anomer), 2.19 (3H, s, -OCOCH3 β anomer)

2.16 (3H, s, -OCOCH3 β anomer).

13C-NMR (CDCl3): δ 170.55 (-OCOCH3), 129.01, 128.24 and 126.20 (Ar), 101.90 (-OCHPh),

93.45 (C-1), 78.59 (C-4), 75.40 (C-3), 69.69 (C-2), 68.77 (C-6), 63.78 (C-5), 58.46 (-OCH3),

20.93 (-OCOCH3).

Experiment 32:

(2-O-Acetyl-4,6-O-benzylidene-3-O-methyl-1-O-α-D-mannopyranosyl)-trichloroacetimi-

date 34

The procedure of Experiment 8 was applied to compound 33 (0.070 g, 0.216 mmol), with the

reaction time increased to 3 hours. TLC (3:2 hexane-ethyl acetate) indicated that the reaction

was completed. Purification by silica flash column chromatography (3:2 hexane-ethyl acetate)

afforded 34 (0.010 g, 10 %) as a colourless oil.

vmax/cm-1

: 3337.85 (N-H), 1750.99 (C=O), 1093.79-1035.10 (C-O)

1H-NMR (CDCl3): δ 8.76 (1H, s, -OC(NH)CCl3), 7.54-7.45 (2H, m, Ar), 7.41-7.33 (3H, m, Ar)

6.24 (1H, d, 3JH-H = 1.48 Hz, H-1α), 5.64 (1H, s, -OCHPh), 5.54 (1H, dd,

3JH-H = 3.39 Hz,

3JH-H =

1.76 Hz, H-2), 4.33 (1H, dd, 2JH-H = 10.32 Hz,

3JH-H = 4.50 Hz H-6a), 4.14-4.01 (2H, m, H-4,5),

3.90-3.82 (2H, m, H-3,6b), 3.51 (3H, s, -OCH3), 2.20 (3H, s, -OCOCH3).

13C-NMR (CDCl3): δ 170.41, 167.78 (-OCOCH3 and -OC(NH)CCl3), 129.10, 128.27 and

126.11 (Ar), 101.82 (-OCHPh), 95.55 (C-1), 77.99 (C-4), 75.77 (C-3), 68.41 (C-6), 67.76 (C-2),

66.32 (C-5), 58.76 (-OCH3), 20.87 (-OCOCH3).

89

Experiment 33:

Attempted synthesis of Methyl 3-O-methyl-α-D-mannopyranoside 35

α-methyl-D-mannose (0.100 g, 0.515 mmol) was dissolved in dry toluene (5 mL). Then

dibutyltin oxide (0.128 g, 0.515 mol) was added. The mixture was stirred and refluxed for 3

hours. TBAI (0.190 g, 0.515 mol) and iodomethane (0.097 mL, 1.54 mol) were added

sequentially. The mixture was heated at 70 ºC and stirred for 72 hours. The solvent was first

evaporated under vacuum. The mixture was dissolved in methanol and filtered. The solvent of

the filtrate was removed under vacuum and the reaction mixture was purified by flash column

chromatography, (Eluent from 12:1 dichloromethane-methanol to 9:1 dichloromethane-

methanol) and interpretation of the obtained 1H-NMR spectrum from the compound revealed

that the reaction did not happen.

Experiment 34:

Attempted synthesis of Methyl 3-O-methyl-α-D-mannopyranoside 35

The procedure of Experiment 10 was applied to α-methyl-D-mannose (0.100 g, 0.515 mmol).

Purification by silica preparative flash column chromatography (Eluent from 12:1

dichloromethane-methanol to 9:1 dichloromethane-methanol) and interpretation of the obtained

1H-NMR spectrum from the compound revealed that the reaction did not happen.

90

Experiment 35:

Methyl 6-O-trityl-α-D-mannopyranoside 36

α-methyl-D-mannose (0.200 g, 1.03 mmol) was dissolved in dry pyridine. Then, TrCl (0.359 g,

1.29 mmol) was added, and the mixture was stirred at room temperature for 24 hours. After 24

hours more quantity of TrCl (0.287 g, 1.03 mmol) and DMAP (0.015 g, 0.124 mmol) were

added. The mixture was stirred at the same temperature for 18 hours. TLC (1:4 hexane-ethyl

acetate) indicated that the reaction was completed. Purification of the reaction crude, by flash

column chromatography (1:4 hexane-ethyl acetate) afforded 36 (0.449 g, 100 %) as a colourless

viscous foam.

vmax/cm-1

: 3405.6 (O-H), 1056.01 (C-O)

1H-NMR (CDCl3): δ 7.47-7.43 (5H, m, Ar), 7.34-7.27 (10H, m, Ar), 4.72 (1H, br s, H-1), 3.92

(1H, br d, 3JH-H = 1.64 Hz, H-2), 3.79 (1H, dd,

2JH-H = 8.89 Hz,

3JH-H = 3.34 Hz, H-3), 3.72 (1H,

t, 3JH-H = 9.08 Hz, H-4), 3.69-3.63 (1H, m, H-5), 3.47 (1H, dd,

3JH-H = 9.77 Hz,

3JH-H = 4.75 Hz,

H-6a), 3.41 (1H, dd, 3JH-H = 9.82 Hz,

3JH-H = 5.39 Hz, H-6b), 3.38 (3H, s, -OCH3).

13C-NMR (CDCl3): 128.59, 128.01 and 127.26 (Ar), 100.54 (C-1), 71.62 (C-3), 70.56 (C-4),

70.23 (C-2), 69.54 (C-5), 64.94 (C-6), 56.01 (-OCH3).

Experiment 36:

Methyl 3-O-methyl-6-O-trityl-α-D-mannopyranoside 37


hexane-ethyl acetate) indicated that the reaction was completed. Purification of the reaction

crude, by flash column chromatography (2:3 hexane-ethyl acetate) afforded 37 (0.140 g, 68 %)

as a yellowish viscous foam.

vmax/cm-1

: 3403.35 (O-H), 1057.30-1023.54 (C-O)

91

1H-NMR (CDCl3): δ 7.48-7.42 (5H, m, Ar), 7.31-7.20 (10H, m, Ar), 4.76 (1H, d,

3JH-H = 1.35

Hz, H-1), 4.02 (1H, br s, H-2), 3.76-3.70 (2H, m, H-4,5), 3.44 (3H, s, -OCH3), 3.43-3.40 (3H,

m, H-3,6a,6b), 3.39 (3H, s,-OCH3).

13C-NMR (CDCl3): δ 128.65, 127.93 and 127.14 (Ar), 100.34 (C-1), 80.89 (C-3), 69.94 and

68.75 (C-4 and C-5), 69.77 (C-2), 64.89 (C-6), 57.34 (-OCH3), 54.94 (-OCH3).

Experiment 37:

1,2,4,6-Tetra-O-acetyl-3-O-methyl-(α/β)-D-mannopyranose 38

Compound 37 (0.140 g, 0.311 mmol) was dissolved in distilled acetic anhydride/acetic

acid/sulfuric acid (105:45:1, v/v/v, 1.2 mL). The mixture was stirred overnight at room

temperature. TLC (2:3 hexane-ethyl acetate) indicated that the reaction was completed. The

mixture was neutralized and washed with an aqueous solution of sodium hydrogen carbonate

(saturated) and extracted with dichloromethane. The organic layer was dried with Na2SO4,

filtered and concentrated. Purification by flash column chromatography, (2:3 hexane-ethyl

acetate) afforded 38 (0.090 g, 80%, α/β > 10:1) as a yellowish oil.

NMR data for the α anomer was in accordance with those described in the literature.[21]

92

93

CHAPTER 5

REFERENCES

94

95

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